Synthetic oligosaccharides for moraxella vaccine

ABSTRACT

The present invention provides synthetic  Moraxella catarrhalis  lipooligosaccharide (LOS)-based oligosaccharides and conjugates containing various  M. catarrhalis  serotype-specific oligosaccharide antigens or various core  M. catarrhalis  oligosaccharide structures or motifs corresponding to one or more of the three major serotypes and/or members within a given serotype. The oligosaccharides may be synthesized by a chemical assembly methodology relying on a limited number of monosaccharide and disaccharide building blocks. The invention further provides  M. catarrhalis  LOS-based immunogenic and immunoprotective compositions and antibodies derived therefrom for diagnosing, treating, and preventing infections caused by  M. catarrhalis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/643,184, filed Jan. 11, 2013, which is a national stage applicationof PCT/US2011/034172, filed Apr. 27, 2011, which claims the benefit ofU.S. Provisional Application No. 61/328,576, filed Apr. 27, 2010. Theentire contents of the aforementioned applications are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to immunogenic and immunoprotectivecompositions and methods for making and using homogenous syntheticMoraxella catarrhalis lipooligosaccharide (LOS)-based oligosaccharides,conjugates, and antibodies derived therefrom.

BACKGROUND

M. catarrhalis is an important human mucosal pathogen that contributesto otitis media in infants and exacerbates conditions such as chronicobstructive pulmonary disease in the elderly. In view of the increasedincidence of M. catarrhalis infection and increased virulence andantibiotic resistance found in modern clinical isolates, there is a needto identify and develop new therapies targeting this pathogen.Currently, there is no M. catarrhalis vaccine approved for human use.

The lipooligosaccharides (LOS) of M. catarrhalis share a high degree ofstructural homology across the three known serotypes (A, B and C),including a common glucose core, indicating the potential for broadbased coverage if an antibody response to a common epitope can beelicited.

The present invention provides a synthetically produced vaccine approachtargeting the LOS cores of M. catarrhalis. Native M. catarrhalis LOSsare composed of branched oligosaccharides anchored to the cell membranevia a KDO₂-lipid A linker. The KDO₂-lipid A glycolipid motif is found inmany species of gram negative bacteria (Edebrink et al., Carbohydr.Res., 295:127-146, 1996).

There is evidence that an antibody response to the LOS is a major partof the natural human immune response and that the anti-LOS antibodiesfrom exposed patients are bactericidal to all the M. catarrhalisserotypes (U.S. Pat. No. 6,685,949). Monoclonal antibodies derived fromanimals exposed to killed M. catarrhalis are able to bind to all threeserotype LOS structures equally well and display bactericidal activitytowards all (Gergova et al., Curr. Microbiol., 54:85, 2007). Theseantibodies are thought to bind to an LOS core structure shared by allthree serotypes. These results suggest that it should be possible toelicit an immunogenic response against all three serotypes using anantigen composed of the M. catarrhalis LOS core or derivative thereof.

LOS preparations isolated and purified from cellular material have beenshown to be lethal to mice (Fomsgaard et al., Infect. Immun., 66:1891,1998). The toxicity of the LOS preparations has been attributed to theKDO₂-lipid A motif, more specifically the lipid A component. Id. Severalgroups have tried to remove the lipid A portion from the isolatedmolecules (Gu et al., Infect. Immun., 66:1891, 1998; Yu et al., Infect.Immun., 73:2790, 2005; Yu et al., Infect. Immun., 75:2974, 2007; U.S.Pat. No. 6,685,949). However, these methods do not completely andconsistently remove the toxic lipid A portion.

The present invention provides synthetic M. catarrhalis LOS cores thatare free of the KDO₂-lipid A moiety and are homogenous. Such syntheticcores of M. catarrhalis LOS provide several benefits for vaccinedevelopment. The present invention allows for the production ofhomogenous antigen compositions at high purity and at robust levelswithout contaminating carbohydrate structures that are an almostinevitable consequence of isolation from biological mixtures.

These synthetic oligosaccharide cores may include a linker toconveniently facilitate formation of conjugate molecules. Thesesynthetic oligosaccharide cores can be modified in ways not possiblewith isolates from natural sources, i.e., by systematically varyinglength and composition, by functionalizing side chains, and bycontrolling the antigen-protein carrier conjugation ratio, etc. Thisenables ready access to minor sequences, deletion sequences, and othervariants on the natural structures that would be difficult or impossibleto obtain from natural sources in high purity and sufficient quantityfor conjugation.

SUMMARY

The present invention provides synthetic oligosaccharides 1a:

where each of R¹ and R² is independently H, a monosaccharide or anoligosaccharide, and X is H or a protecting group. Oligosaccharides 1ainclude core oligosaccharides shared by the three M. catarrhalisserotypes.

The present invention further provides antigens 1b:

where each of R¹ and R² is independently H, a monosaccharide or aoligosaccharide; L is a linker; and Y is H or a carrier. Preferably, theantigens are free of endotoxins.

The present invention also includes compositions comprising an antigen1b and a pharmaceutically acceptable vehicle. Preferably the compositioncontains a single antigen or a known, defined mixture of antigens.

The invention further provides vaccine compositions, includingimmunogenic and immunoprotective compositions, comprising antigen 1b anda pharmaceutically acceptable vehicle. These vaccine compositions canoptionally include a pharmaceutically acceptable adjuvant. Preferably,the vaccine compositions are endotoxin-free. The vaccine compositionscan be mono-, di-, tri- or tetravalent.

The invention further provides a method for synthetically formingoligosaccharides 1a and antigens 1b.

The invention further provides methods for diagnosing, treating, andpreventing infections caused by M. catarrhalis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts LOS structures shared by the three M. catarrhalisserotypes, which contain a common, highly branched glucose core and aKDO₂-lipid A motif at the reducing end. Structural variance can be foundin the α and β chains. While all three serotypes are comprised ofglucose and galactose residues, the A and C serotypes also contain anN-acetyl glucosamine in the β chain.

FIG. 2 depicts the α and β chains of the naturally occurring M.catarrhalis LOS structures shown in FIG. 1. The shaded entries are themost common sequence found in each serotype; the other entriescorrespond to sequences found as lesser components in LOS fractions andto exemplary core oligosaccharides or motifs corresponding to one ormore of the three major serotypes and/or members within a givenserotype.

FIG. 3 depicts common intermediates used for synthesizing theoligosaccharide antigens of the present invention.

FIGS. 4A and 4B depict reaction schemes for forming monosaccharidebuilding blocks as described in Example 1, including allyl4,6-O-benzylidene-2-O-pivaloyl-α-D-glucopyranoside 1 (FIG. 4A); and2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranosyl trichloroacetimidate 2(FIG. 4B).

FIGS. 5A and 5B depict reactions schemes for synthesizing a tetramercore 5 (FIG. 5A); and conjugates 49, 50 thereof (FIG. 5B) as describedin Example 2.

FIGS. 6A and 6B depict reaction schemes for synthesizing a heptamer core46 (FIG. 6A); and conjugates 47, 48 thereof (FIG. 6B) as described inExample 3.

FIGS. 7A and 7B depict reaction schemes for synthesizing a Serotype Aoctasaccharide 27 (FIG. 7A); and conjugates 28, 29 thereof (FIG. 7B) asdescribed in Example 4.

FIGS. 8A and 8B depict reaction schemes for synthesizing a Serotype B7hexasaccharide 8 (FIG. 8A); and conjugates 9, 41 thereof (FIG. 8B) asdescribed in Example 5.

FIG. 9 depicts a reaction scheme for synthesizing a Serotype B9octasaccharide 11 and conjugates 12, 40 thereof as described in Example6.

FIGS. 10A and 10B depict a reaction scheme for synthesizing a SerotypeB11 decasaccharide 20 and conjugates 21, 22 thereof as described inExample 7.

FIGS. 11A and 11B depict a reaction scheme for synthesizing a SerotypeC11 decasaccharide 37 and conjugates 38, 39 thereof as described inExample 8.

FIG. 12 depicts a reaction scheme for synthesizing a Serotype C11heptamer substructure 52 and conjugates 42, 53 thereof as described inExample 9.

FIGS. 13A-13G depict ELISA results from Example 10, showing IgG antibodytiters as a function of antibody-antigen complex absorption (OD₄₅₀) at 3serum dilutions of immune sera obtained from 3 succesive bleeds(pre-immune′ 1^(st) bleed, and final bleed) in rabbits (n=2) immunizedwith antigen-KLH conjugates corresponding to (A) Serotype A-KLH 28; (B)Serotype B7 hexasaccharide core-KLH 9; (C) Serotype B9-KLH 12; (D)Serotype B11-KLH 21; (E) Serotype C11 decamer-KLH 38; (F) SerotypeC11-heptamer-KLH 53; (G) heptamer core-KLH 47; and (H) tetramer core-KLH49. In each case, the antisera were incubated on ELISA plates adsorbedwith their corresponding BSA conjugate, specifically: (A) Serotype A-BSA29; (B) Serotype B7-BSA 41; (C) Serotype B9-BSA 40; (D) Serotype B11-BSA22; (E) Serotype C11-decamer-BSA 39; (F) Serotype C11-heptamer-BSA 42;(G) heptamer core-BSA 48; and (H) tetramer core-BSA 50.

FIGS. 14A-14D depict specificity and cross-reactivity of antisera todifferent synthetic Moraxella LOS oligosaccharides by ELISA as describedin Example 11. Antisera from rabbits immunized with the indicatedantigen-KLH conjugates corresponding to (left to right) KLH alone,tetramer core-KLH 49, hexamer core (B7)-KLH 9, heptamer core-KLH 47,Serotype B11-KLH 21, Serotype A-KLH 28, and Serotype C11-decamer-KLH 38were incubated with ELISA plates adsorbed with antigen-BSA conjugates,including (A) Serotype A-BSA 29; (B) Serotype B11-BSA 22; (C) SerotypeC11-decamer-BSA 39; and (D) Serotype C11-heptamer-BSA 42. Results areshown as a function of antibody-antigen complex absorption (OD₄₅₀) atthe indicated serum dilutions.

DETAILED DESCRIPTION Definitions

In order to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided.

Units, prefixes, and symbols may be denoted in their SI accepted form.Numeric ranges recited herein are inclusive of the numbers defining therange and include and are supportive of each integer within the definedrange. Unless otherwise noted, the terms “a” or “an” are to be construedas meaning “at least one of.” The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described. All documents, or portions of documents, citedin this application, including but not limited to patents, patentapplications, articles, books, and treatises, are hereby expresslyincorporated by reference in their entirety for any purpose.

As used herein, “oligosaccharide” refers to a compound containing two ormore monosaccharide units. Oligosaccharides are considered to have areducing end and a non-reducing end, whether or not the monosaccharideunit at the reducing end is in fact a reducing sugar. In accordance withaccepted nomenclature, oligosaccharides are depicted herein with thenon-reducing end on the left and the reducing end on the right. Alloligosaccharides described herein are described with the name orabbreviation for the non-reducing monosaccharide (e.g., Gal), precededby the configuration of the glycosidic bond (α or β), the ring bond, thering position of the reducing monosaccharide involved in the bond, andthen the name or abbreviation of the reducing monosaccharide (e.g.,GlcNAc). The linkage between two sugars may be expressed, for example,as 2,3, 2→3, or 2-3. Each monosaccharide is a pyranose or furanose.

As used herein, “monosaccharide” or “monosaccharide unit” refers to asingle sugar residue in an oligosaccharide, including derivativestherefrom. Within the context of an oligosaccharide, an individualmonomer unit is a monosaccharide which is (or can be) bound through ahydroxyl group to another monosaccharide.

As used herein, “endotoxin-free” refers to an oligosaccharide that doesnot contain endotoxins or endotoxin components normally present inisolated bacterial carbohydrates and polysaccharides.

As used herein, “synthetic” refers to material which is substantially oressentially free from components, such as endotoxins, glycolipids,unrelated oligosaccharides, etc., which normally accompany a compoundwhen it is isolated. Typically, synthetic compounds are at least about90% pure, usually at least about 95%, and preferably at least about 99%pure. Purity can be indicated by a number of means well known in theart. Preferably, purity is measured by HPLC. The identity of thesynthetic material can be determined by mass spectroscopy and/or NMRspectroscopy.

As used herein the term “linker” refers to either a bond or a moietywhich at one end exhibits a grouping able to enter into a covalentbonding with a reactive functional group of the carrier, e.g. an amino,thiol, or carboxyl group, and at the other end a grouping likewise ableto enter into a covalent bonding with a hydroxyl group or an amino groupof an oligosaccharide according to the present invention. Between thetwo functional groups of the linker molecule there is a biocompatiblebridging molecule of suitable length, e.g. substituted or unsubstitutedheteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkyleneglycol groups. Linkers preferably include a substituted or unsubstituted(C₁-C₁₀) alkylene group or an substituted or unsubstituted (C₂-C₁₀)alkenylene group.

As used herein, the term “carrier” refers to a protein, peptide, lipid,polymer, dendrimer, virosome, virus-like particle (VLP), or combinationthereof, which is coupled to the oligosaccharide to enhance theimmunogenicity of the resulting oligosaccharide-carrier conjugate to agreater degree than the oligosaccharide alone.

As used herein, “protein carrier” refers to a protein, peptide orfragment thereof, which is coupled or conjugated to an oligosaccharideto enhance the immunogenicity of the resulting oligosaccharide-proteincarrier conjugate to a greater degree than the oligosaccharide alone.For example, when used as a carrier, the protein carrier may serve as aT-dependent antigen which can activate and recruit T-cells and therebyaugment T-cell dependent antibody production.

As used herein, “conjugated” refers to a chemical linkage, eithercovalent or non-covalent, that proximally associates an oligosaccharidewith a carrier so that the oligosaccharide conjugate has increasedimmunogenicity relative to an unconjugated oligosaccharide.

As used herein, “conjugate” refers to an oligosaccharide chemicallycoupled to a carrier through a linker and/or a cross-linking agent.

As used herein, “passive immunity” refers to the administration ofantibodies to a subject, whereby the antibodies are produced in adifferent subject (including subjects of the same and different species)such that the antibodies attach to the surface of the bacteria and causethe bacteria to be phagocytosed or killed.

As used herein, “protective immunity” means that a vaccine orimmunization schedule that is administered to a animal induces an immuneresponse that prevents, retards the development of, or reduces theseverity of a disease that is caused by a pathogen or diminishes oraltogether eliminates the symptoms of the disease. Protective immunitymay be predicted based on the ability of serum antibody to activatecomplement-mediated bactericidal activity or confer passive protectionagainst a bacterial infection in a suitable animal challenge model.

As used herein, “immunoprotective composition” refers to a compositionformulated to provide protective immunity in a host.

As used herein, “in a sufficient amount to elicit an immune response” or“in an effective amount to stimulate an immune response” (e.g., toepitopes present in a preparation) means that there is a detectabledifference between an immune response indicator measured before andafter administration of a particular antigen preparation. Immuneresponse indicators include but are not limited to: antibody titer orspecificity, as detected by an assay such as enzyme-linked immunoassay(ELISA), bactericidal assay (e.g., to detect serum bactericidalantibodies), flow cytometry, immunoprecipitation, Ouchter-Lowryimmunodiffusion; binding detection assays of, for example, spot, Westernblot or antigen arrays; cytotoxicity assays, and the like.

As used herein, “antibody” encompasses polyclonal and monoclonalantibody preparations, as well as preparations including hybridantibodies, altered antibodies, F(ab′)² fragments, F(ab) molecules, Fvfragments, single chain fragment variable displayed on phage (scFv),single domain antibodies, chimeric antibodies, humanized antibodies, andfunctional fragments thereof which exhibit immunological bindingproperties of the parent antibody molecule.

As used herein, “monoclonal antibody” refers to an antibody compositionhaving a homogeneous antibody population. The term is not limited by themanner in which it is made. The term encompasses whole immunoglobulinmolecules, as well as Fab molecules, F(ab′)₂ fragments, Fv fragments,single chain fragment variable displayed on phage (scFv), and othermolecules that exhibit immunological binding properties of the parentmonoclonal antibody molecule.

As used herein, “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to an oligosaccharide, protein orpeptide, refers to a binding reaction which is based on and/or isprobative of the presence of the antigen in a sample which may alsoinclude a heterogeneous population of other molecules. Thus, underdesignated immunoassay conditions, the specified antibody or antibodiesbind(s) to a particular antigen or antigens in a sample and does notbind in a significant amount to other molecules present in the sample.Specific binding to an antibody under such conditions may require anantibody or antiserum that is selected for its specificity for aparticular antigen or antigens.

As used herein, “antigen” refers to any substance that may bespecifically bound by an antibody molecule.

As used herein, “immunogen” and “immunogenic composition” refer to anantigenic composition capable of initiating lymphocyte activationresulting in an antigen-specific immune response.

As used herein, “epitope” refers to a site on an antigen to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite.” B cell epitope sites on proteins, oligosaccharides, or otherbiopolymers may be composed of moieties from different parts of themacromolecule that have been brought together by folding. Epitopes ofthis kind are referred to as conformational or discontinuous epitopes,since the site is composed of segments the polymer that arediscontinuous in the linear sequence but are continuous in the foldedconformation(s). Epitopes that are composed of single segments ofbiopolymers or other molecules are termed continuous or linear epitopes.T cell epitopes are generally restricted to linear peptides. Antibodiesthat recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen.

The term Ac means acetyl (—C(O)CH₃).

The term TBS means tert-butyldimethylsilyl.

The term Troc means 2,2,2-trichloroethoxycarbonyl.

The term TCl means trichloroacetimidate.

The term Phth means phthaloyl.

The term TFA means trifluoroacetate.

The term TCA means trichloroacetate.

The term Cbz means benzyloxycarbonyl.

The term Bz means benzoyl.

The term Bn means benzyl.

The term TES means triethylsilyl.

The term TBDPS means tert-butyldiphenylsilyl.

The term MCA means monochloracetate.

The term Lev means levulinoyl.

The term ADMB means 4-O-acetyl 12,2 dimethylbutanoate.

The term Tr means triphenylmethyl.

The term DMT means dimethoxytrityl.

The term FMOC means 9-fluorenylmethyl carbonate.

The term Alloc means Allyloxycarbonyl.

The term Nap means napthyl.

The term SEt means thioethyl.

The term SPh means thiophenyl.

The term STol means thiotolyl.

The term SAdm means thioadamantyl.

Synthetic Oligosaccharides

The present invention provides compositions and methods for chemicallysynthesizing antigenic structures corresponding to the M. catarrhalislipooligosaccharide (LOS), a major surface component of the outermembrane. Greater than 95% of isolated M. catarrhalis strains possessone of three LOS serotypes: A (61%), B (29%) and C (5%; Vaneechoutte etal., J. Clin. Microbiol., 28:182, 1990). The LOS structures of all threeserotypes have been thoroughly characterized and are composed of abranched oligosaccharide anchored to the cell membrane via a KDO₂-lipidA moiety, a common glycolipid motif found in many species of gramnegative bacteria (Edebrink et al., Carbohydr. Res., 257:269, 1994;Edebrink et al., Carbohydr. Res., 295:127, 1996; Edebrink et al.,Carbohydr. Res., 266:237, 1995). The oligosaccharide portion from allthree of the characterized LOS structure serotypes contains a common,highly branched glucose core (FIG. 1). The structural variance observedamong the three serotypes is found in the composition of the α and βchains (FIGS. 1 and 2; Braun et al., Vaccine, 22:898, 2004). The threeserotypes all are comprised of glucose and galactose residues and the Aand C serotypes also contain an N-acetyl glucosamine in the β chain.Analytical characterization shows that each serotype generates a fulllength, primary LOS structure, defined as A, B11 and C11. Additionalcharacterization of the B and C M. catarrhalis serotypes reveals thatdifferent strains of these bacteria also display additional forms of theB and C oligosaccharides, representing partial deletion sequences of theprimary serotype antigen (FIG. 2). Of the three serotypes, the Bserotype shows the greatest natural variability; the full lengthsequence B11 is thought to be the primary virulence factor while theother B sequences are believed to be expressed at different points inthe bacterial life cycle (Braun et al., Vaccine, 22:898, 2004).

FIG. 2 depicts the composition of a and β chains of the naturallyoccurring M. catarrhalis LOS structures. The shaded entries representthe most common sequence found in each serotype; the other entriesinclude deletion sequences found as lesser components in LOS fractions(Braun et al., Vaccine, 22:898, 2004). In its natural presentation, thelipid portion is buried in the bacterial cell membrane, presenting theoligosaccharide region to the surrounding environment.

In one aspect, the present invention provides oligosaccharides 1a:

where each of R¹ and R² is independently H, a monosaccharide or aoligosaccharide, and X is H or a protecting group.

The present invention further provides antigens 1b:

where each of R¹ and R² is independently H, a monosaccharide or aoligosaccharide; L is a linker; and Y is H or a carrier.

In compounds 1a and 1b, oligosaccharides may include one or moremonosaccharide units linked to one another through one or more α- and/orβ-glycosidic bonds. Preferably, the oligosaccharides will includemonosaccharides and glycosidic linkages naturally found in M.catarrhalis LOS structures, generally in 1-2 or 1-4 connectivities. Theinvention further contemplates other connectivities, such as 1-3 and1-6, especially where the oligosaccharide design is extended beyondnaturally M. catarrhalis LOS structures.

When either of R¹ or R² is an oligosaccharide, it may contain between 1to about 6, preferably up to about 4, monosaccharide units. Theinvention contemplates inclusion of natural and modified monosaccharideunits, such as D-mannose, D-galactose, D-glucosamine, D-fucose, andsialic acid, especially where the oligosaccharide design is extendedbeyond naturally M. catarrhalis LOS structures.

In compounds 1a and 1b, preferred monosaccharides include Gal(galactosyl); GalNAc (N-acetylgalactosaminyl); Glc (glucosyl); GlcNAc(N-acetylglucosaminyl); sialic acid (Neu5Ac).

In compounds 1a and 1b, preferred oligosaccharides for R¹ and R² aredi-, tri-, and tetra-saccharides. Preferred oligosaccharides contain oneor more units of Gal, GalNAc, Glc, and GlcNAc. The units are preferablyconnected via 1-4 bonds.

Preferred oligosaccharides 1a according to the present invention havethe R¹ and R² values in Table 1.

TABLE 1 preferred oligosaccharides 1a 1a

Compound # R1 R2 56 αGlc(1-2) H  8 αGlc(1-2) 70 βGal(1-4)αGlc(1-2) 71αGal(1-4)βGal(1-4)αGlc(1-2) 52 αGlcNAc(1-2) 54 βGal(1-4)αGlcNAc(1-2) 72αGal(1-4)βGal(1-4)αGlcNAc(1-2)  5 H H 72 αGlc(1-2) 74 βGal(1-4)αGlc(1-2)75 αGal(1-4)βGal(1-4)αGlc(1-2) 76 αGlcNAc(1-2) 77 βGal(1-4)αGlcNAc(1-2)78 αGal(1-4)βGal(1-4)αGlcNAc(1-2) 79 βGal(1-4)αGlc(1-2) H 80 αGlc(1-2)11 βGal(1-4)αGlc(1-2) 81 αGal(1-4)βGal(1-4)αGlc(1-2) 82 αGlcNAc(1-2) 83βGal(1-4)αGlcNAc(1-2) 84 αGal(1-4)βGal(1-4)αGlcNAc(1-2) 46αGal(1-4)βGal(1-4) H 85 αGlc(1-2) αGlc(1-2) 86 βGal(1-4)αGlc(1-2) 20αGal(1-4)βGal(1-4)αGlc(1-2) 27 αGlcNAc(1-2) 87 βGal(1-4)αGlcNAc(1-2) 37αGal(1-4)βGal(1-4)αGlcNAc(1-2)

In Table 1, X is preferably H. In another embodiment, X is preferably aprotecting group.

In another aspect, the present invention provides LOS antigens comprisedof core oligosaccharide structures or motifs corresponding to one ormore of the three major serotypes, members within a given serotype, andindividual serotype subtypes as depicted in Table 1. In the followingembodiments, L is exemplified as an alkylene thiol group, where p is aninteger from 1 to 20, preferably between 1 and 8. In any of theseembodiments, the linker shown (i.e., the alkylene thiol group) could bereplaced with any other suitable linker as described herein. Thefollowing embodiments are identified elsewhere by Formula numberscorresponding to oligosaccharides of Formula 1a where the linker is—(CH₂)₃—S—, however, the structures are depicted as having generic thiollinkers.

Thus, in one embodiment, the invention provides an tetrasaccharide 5:

In another embodiment, the invention provides an pentasaccharide 56:

In another embodiment, the invention provides a heptasaccharide 46:

In another embodiment, the invention provides a hexsaccharide 52:

In another embodiment, the invention provides an octasaccharide 27:

In another embodiment, the invention provides a hexasaccharide 8:

In a further embodiment, the invention provides an octasaccharide 11:

In a further embodiment, the invention provides a decasaccharide 20:

In another embodiment, the invention provides a heptasaccharide 54:

In a further embodiment, the invention provides a decasaccharide 37:

In a further embodiment, the invention provides a heptasaccharide 40:

It should be recognized, that the present invention contemplates andprovides sufficient guidance below for modifying any of theabove-described thiol products with different linkers and/or spacers,and to make LOS structures directed to any of the oligosaccharidesequences listed in FIG. 2, including any subsequence combinationsderived therefrom, or indeed, any M. catarrhalis LOS structure for thatmatter.

In a further aspect, the invention provides polyvalent LOS antigencombinations (and conjugates thereof) representing pluralities of any ofthe different oligosaccharides described in Table 1 or FIG. 2, forexample.

The compositions can be mono-, di-, tri- or tetra-valent, containingantigens against the same serotype (i.e. two antigens to serotype A) ordifferent serotypes. For example, the following compositions containingpolyvalent antigen combinations aganst different serotypes arecontemplated below in Table 2:

TABLE 2 combination vaccines Vaccine Serotype No. A B C core CV1 0 0 1 1CV2 0 1 0 1 CV3 0 1 1 0 CV4 0 1 1 1 CV5 1 0 0 1 CV6 1 0 1 0 CV7 1 0 1 1CV8 1 1 0 0 CV9 1 1 0 1 CV10 1 1 1 0 CV11 1 1 1 1 1 = present; 0 =absent

Compositions of the present invention include LOS-oligosaccharidestructures defined by Formulas 1a and 1b, which may include a linker (L)and may optionally contain a carrier (Y is H or a carrier).

Suitable linkers comprise at one end a grouping able to enter into acovalent bonding with a reactive functional group of the carrier, e.g.an amino, thiol, or carboxyl group, and at the other end a groupinglikewise able to enter into a covalent bonding with a hydroxyl group ofan oligosaccharide according to the present invention. Between the twofunctional groups of the linker molecule there is a biocompatiblebridging molecule of suitable length, e.g. substituted or unsubstitutedheteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkyleneglycol groups. Linkers preferably include substituted or unsubstitutedalkylene or alkenylene groups containing 1-10 carbon atoms.

Linkers able to react with thiol groups on the carrier are, for example,maleimide and carboxyl groups; preferred groupings able to react withaldehyde or carboxyl groups are, for example, amino or thiol groups.Preferred covalent attachments between linkers and carriers includethioethers from reaction of a thiol with an α-halo carbonyl or α-halonitrile, including reactions of thiols with maleimide; hydrazides fromreaction of a hydrazide or hydrazine with an activated carbonyl group(e.g. activated NHS-ester or acid halide); triazoles from reaction of anazide with an alkyne (e.g. via “click chemistry”); and oximes fromreaction of a hydroxylamine and an aldehyde or ketone as disclosed, forexample, in Lees et al., Vaccine, 24:716, 2006. Although amine-basedconjugation chemistries could be used in principle for coupling linkersand/or spacers to the oligosaccharides described herein, theseapproaches would typically sacrifice uniformity inasmuch as theoligosaccharides of the present invention typically contain a pluralityof amines bonded to second carbon of the respective monosaccharideunits.

Further suitable linker molecules are known to skilled workers andcommercially available or can be designed as required and depending onthe functional groups present and can be prepared by known methods.

Suitable carriers are known in the art (See e.g., Remington'sPharmaceutical Sciences (18th ed., Mack Easton, Pa. (1990)) and mayinclude, for example, proteins, peptides, lipids, polymers, dendrimers,virosomes, virus-like particles (VLPs), or combinations thereof, whichby themselves may not display particular antigenic properties, but cansupport immunogenic reaction of a host to the oligosaccharides of thepresent invention (antigens) displayed at the surface of the carrier(s).

Preferably, the carrier is a protein carrier, including but are notlimited to, bacterial toxoids, toxins, exotoxins, and nontoxicderivatives thereof, such as tetanus toxoid, tetanus toxin Fragment C,diphtheria toxoid, CRM (a nontoxic diphtheria toxin mutant) such as CRM197, cholera toxoid, Staphylococcus aureus exotoxins or toxoids,Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosaexotoxin A, including recombinantly produced, genetically detoxifiedvariants thereof; bacterial outer membrane proteins, such as Neisseriameningitidis serotype B outer membrane protein complex (OMPC), outermembrane class 3 porin (rPorB) and other porins; keyhole limpethemocyanine (KLH), hepatitis B virus core protein, thyroglobulin,albumins, such as bovine serum albumin (BSA), human serum albumin (HSA),and ovalbumin; pneumococcal surface protein A (PspA), pneumococcaladhesin protein (PsaA); purified protein derivative of tuberculin (PPD);transferrin binding proteins, polyamino acids, such aspoly(lysine:glutamic acid); peptidyl agonists of TLR-5 (e.g. flagellinof motile bacteria like Listeria); and derivatives and/or combinationsof the above carriers. Preferred carriers for use in humans includetetanus toxoid, CRM 197, and OMPC.

Depending on the type of bonding between the linker and the carrier, andthe structural nature of the carrier and oligosaccharide, a carrier maydisplay on average, for example, 1 to 500, 1 to 100, 1 to 20, or 3 to 9oligosaccharide units on its surface.

Methods for attaching an oligosaccharide to a carrier, such as a carrierprotein are conventional, and a skilled practitioner can createconjugates in accordance with the present invention using conventionalmethods. Guidance is also available in various disclosures, including,for example, U.S. Pat. Nos. 4,356,170; 4,619,828; 5,153,312; 5,422,427;and 5,445,817; and in various print and online Pierce proteincross-linking guides and catalogs (Thermo Fisher, Rockford, Ill.).

In one embodiment, the carbohydrate antigens of the present inventionare conjugated to CRM 197, a commercially available protein carrier usedin a number of FDA approved vaccines. CRM-conjugates have the advantageof being easier to synthesize, purify and characterize than other FDAapproved carriers such as OMPC. Carbohydrate antigens may be conjugatedto CRM via thiol-bromoacetyl conjugation chemistry. CRM activation maybe achieved by reacting the lysine side chains with the NHS ester ofbromoacetic acid using standard conditions as previously described inU.S. Pat. Appl. Publ. 2007-0134762, the disclosures of which areincorporated by reference herein. CRM may be functionalized with 10-20bromoacetyl groups per protein (n=10-20) prior to conjugation.Conjugation may be performed at pH=9 to avoid aggregation of CRM.Careful monitoring of pH must be employed to ensure complete CRMreaction with NHS-bromoacetate while minimizing background hydrolysis ofCRM. Activated CRM may be purified by size exclusion chromatographyprior to conjugation. Antigen-CRM conjugates may be synthesized byreacting thiol-terminated carbohydrate antigens withbromoacetamide-activated CRM.

CRM conjugates may be purified via size exclusion chromatography toremove and recover any unreacted carbohydrate. MBTH (specific for GlcNAcresidues) and Bradford assays may be used to determinecarbohydrate:protein ratio and protein content, respectively, aspreviously described (Manzi et al., Curr. Prot. Mol. Biol., section17.9.1 (Suppl. 32), 1995. In preferred embodiments, a minimumcarbohydrate content of about 10% by weight for each conjugate may begenerated. Typically, a conjugate may include about 3-20 antigens perprotein carrier.

In another embodiment, carbohydrate antigens may be conjugated to one ormore carriers suitable for development of diagnostic assays, includingELISAs and microarrays. Exemplary carriers for use in such assaysinclude bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH),biotin, a label, a glass slide or a gold surface. By way of example,synthetic carbohydrate antigens may be conjugated to BSA by athiol-maleimide coupling procedure (FIG. 5B). Maleimide-BSA contains15-20 maleimide groups per protein (n=15-20). Accordingly,oligosaccharide antigens may be conjugated to maleimide functionalizedBSA, whereby a 20-fold molar excess of the antigen is reacted withcommercially available Imject maleimide BSA (Pierce) in maleimideconjugation buffer (Pierce). Conjugation may be performed at pH=7.2 toavoid hydrolysis of the maleimide group during conjugation.

BSA conjugates may be purified via size exclusion chromatography toremove and recover any unreacted carbohydrate. Characterization via thephenol-sulfuric acid and Bradford assays may be performed along withMALDI-MS to provide information on the carbohydrate content and valencyof the conjugates. In preferred embodiments, conjugates will contain aminimum carbohydrate content of about 10% by weight per BSA conjugateand >8 antigen copies per conjugate.

Compositions and methods for synthesis of the above describedLOS-oligosaccharides and conjugates thereof, including others describedin Table 1 and FIG. 2 as further described in Examples 1 to 9 below.

Methods for Synthesizing LOS-Oligosaccharide Structures

In a further aspect, the present invention provides a method forassembling synthetic homogenous LOS-oligosaccharide structures from M.catarrhalis, including those described above from monosaccharide anddisaccharide building blocks.

Thus, in one aspect, the invention provides a monosaccharide buildingblocks of Formulas 1 and 2:

A method for synthesizing the building block 1 is described in Example 1below. A method for synthesizing the building block 2 is described inExample 2 below. Building blocks 1 and 2 are used to generate adisaccharide building block of Formula 3:

Disaccharide building block 3 may be combined with the monosaccharidebuilding block of Formula 2 to produce a tetrasaccharide of Formula 4reflecting a core structure present in all M. catarrhalis LOSstructures:

Other common building blocks are shown in FIG. 3 and an be used tosynthesize core structures 4 and 15. For example, mono and disaccharidebuilding blocks 1, 6, 16, 33, 51, 54, 55, 56, and 59 can be used incombination with the trisaccharide core 15 and the tetrasaccharide core4 can enable production of virtually any M. catarrhalis LOS structure.

The tetrasaccharide core 4 may be elaborated to larger fragments of orcomplete oligosaccharides from M. catarrhalis using an assembly strategysuch as that shown in FIG. 3. The alpha- and beta chains may be addedsequentially or in tandem, and may be the same or different, dependingon the desired target molecule. Elaboration may be accomplished usingmonosaccharide building blocks or di-, tri- or tetrasaccharide buildingblocks, such as those disclosed in steps A and B of FIG. 3.

Compositions and methods for synthesis of the above describedLOS-oligosaccharides and conjugates thereof, including others describedin Table 1 or FIG. 2 are described in Examples 1 to 9 below. Protectinggroups employed in the synthesis of LOS-oligosaccharides may includethose customarily considered in sugar chemistry, for example thosementioned in “Protective Groups in Organic Synthesis”, 3^(rd) edition,T. W. Greene and P. G. M. Wuts (Ed.), John Wiley and Sons, New York,1999.

In one embodiment, the present invention provides A method of synthezinga compound of the formula 90:

comprising contacting a first intermediate of the formula:

with a second intermediate of the formula:

where R⁷ is a Bn or is a monosaccharide or oligosaccharide; R⁸ is a Bnor is a monosaccharide or oligosaccharide; and R⁹ is a protecting groupor linker consisting of —CH₂CH═CH₂, —CH₂CCH, pentenyl, alkenylene,oligoalkyl thiol.

In some embodiments the compound 90 is either

Immunogenic and Immunoprotective Compositions and Methods of their Use

In another aspect, the present invention provides immunogenic andimmunoprotective compositions containing LOS oligosaccharides or LOSoligosaccharide-protein carrier conjugates for inducing an immuneresponse to LOS antigens. The immunogenic compositions may include oneor more adjuvants, as well as pharmaceutically acceptable vehiclessuitable for administration to an animal or individual. An immunogenicor immunoprotective composition will include a “sufficient amount” or“an immunologically effective amount” of a oligosaccharide-proteincarrier conjugate according to the present invention, as well as any ofthe above mentioned components, for purposes of generating an immuneresponse or providing protective immunity, as further defined herein.

In one embodiment, the invention provides an immunogenic compositioncomprising one or more LOS oligosaccharide(s) 1a or LOSoligosaccharide-protein carrier conjugate(s) 1b suitable for inducing animmune response against M. catarrhalis.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a LOS oligosaccharide(s) 1a or LOSoligosaccharide-protein carrier conjugate 1b formulated as a vaccine forprotection against M. catarrhalis infections.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising an oligosaccharide-protein carrier conjugate 1bformulated as a vaccine for protecting against one or more M.catarrhalis serotypes as described herein.

In a further embodiment, the invention provides a pharmaceuticalcomposition comprising an antibody and a physiologically acceptablevehicle for use in a method for providing passive immunity or treatmentagainst one or more M. catarrhalis serotypes. More particularly, theinvention provides an antibody preparation against one or moreLOS-oligosaccharide conjugate 1b compositions in accordance with thepresent invention. The antibody preparation may include any member fromthe group consisting of polyclonal antibody, monoclonal antibody, mousemonoclonal IgG antibody, humanized antibody, chimeric antibody, fragmentthereof, or combination thereof. The invention further contemplates ahybridoma cell producing a monoclonal antibody directed against any ofthe LOS-oligosaccharide described herein.

Administration of oligosaccharides or oligosaccharide-protein carrierconjugates or antibodies thereto may be carried out by any suitablemeans, including by parenteral administration (e.g., intravenously,subcutaneously, intradermally, or intramuscularly); by topicaladministration, of for example, antibodies to an airway surface; by oraladministration; by in ovo injection in birds, for example, and the like.

In specific embodiments, each immunogenic or immunoprotectivecomposition includes one or more oligosaccharide(s) according to Formula1a or 1b or conjugates thereof in a pharmaceutically acceptable vehicleor diluent forming a substantially aqueous mixture. In preferredembodiments, the immunogenic or immunoprotective compositions includesone or more oligosaccharide-protein carrier conjugates(s) in conjunctionwith one or more pharmaceutically acceptable adjuvant(s), vehiclesand/or protein carriers suitable for administration to an animal orindividual.

Adjuvants

An oligosaccharide-protein carrier conjugate composition may furtherinclude one or more immunologic adjuvant(s). An immunologic adjuvant isa compound that, when combined with an antigen, increases the immuneresponse to the antigen as compared to the response induced by theantigen alone so that less antigen can be used to achieve a similarresponse. For example, an adjuvant may augment humoral immune responses,cell-mediated immune responses, or both.

Those of skill in the art will appreciate that the terms “adjuvant,” and“carrier,” can overlap to a significant extent. For example, a substancewhich acts as an “adjuvant” may also be a “carrier,” and certain othersubstances normally thought of as “carriers,” for example, may alsofunction as an “adjuvant.” Accordingly, a substance which may increasethe immunogenicity of the synthetic oligosaccharide or carrierassociated therewith is a potential adjuvant. As used herein, a carrieris generally used in the context of a more directed site-specificconjugation to an oligosaccharide of the present invention, whereby anadjuvant is generally used in a less specific or more generalizedstructural association therewith.

Exemplary adjuvants and/or adjuvant combinations may be selected fromthe group consisting of mineral salts, including aluminum salts, such asaluminum phosphate and aluminum hydroxide (alum) (e.g., Alhydrogel™,Superfos, Denmark) and calcium phosphate; RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion, whereby any of the 3 components MPL, TDM or CWS may also beused alone or combined 2 by 2; toll-like receptor (TLR) agonists,including, for example, agonists of TLR-1 (e.g. tri-acyl lipopeptides);agonists of TLR-2 [e.g. peptidoglycan of gram-positive bacteria likestreptococci and staphylococci; lipoteichoic acid]; agonists of TLR-3(e.g. double-stranded RNA and their analogs such as poly 1:C); agonistsof TLR-4 (e.g. lipopolysaccharide (endotoxin) of gram-negative bacterialike Salmonella and E. coli); agonists of TLR-5 (e.g. flagellin ofmotile bacteria like Listeria); agonists of TLR-6 (e.g. with TLR-2peptidoglycan and certain lipids (diacyl lipopeptides)); agonists ofTLR-7 (e.g. single-stranded RNA (ssRNA) genomes of such viruses asinfluenza, measles, and mumps; and small synthetic guanosine-baseantiviral molecules like loxoribine and ssRNA and their analogs);agonists of TLR-8 (e.g. binds ssRNA); agonists of TLR-9 (e.g.unmethylated CpG of the DNA of the pathogen and their analogs; agonistsof TLR-10 (function not defined) and TLR-11—(e.g. binds proteinsexpressed by several infectious protozoans (Apicomplexa), specifictoll-like receptor agonists include monophosphoryl lipid A (MPL®), 3De-O-acylated monophosphoryl lipid A (3 D-MPL), OM-174 (E. coli lipid Aderivative); OM triacyl lipid A derivative, and other MPL- or lipidA-based formulations and combinations thereof, including MPC-SE, RC-529(Dynavax Technologies), AS01 (liposomes+MPL+QS21), AS02 (oil-in-waterPL+QS-21), and AS04 (Alum+MPL)(GlaxoSmith Kline, Pa.),CpG-oligodeoxynucleotides (ODNs) containing immunostimulatory CpGmotifs, double-stranded RNA, polyinosinic:polycytidylic acid (poly I:C),and other oligonucleotides or polynucleotides optionally encapsulated inliposomes; oil-in-water emulsions, including AS03 (GlaxoSmith Kline,Pa.), MF-59 (microfluidized detergent stabilized squalene oil-in-wateremulsion; Novartis), and Montanide ISA-51 VG (stabilized water-in-oilemulsion) and Montanide ISA-720 (stabilized water/squalene; SeppicPharmaceuticals, Fairfield, N.J.); cholera toxin B subunit; saponins,such as Quil A or QS21, an HPLC purified non-toxic fraction derived fromthe bark of Quillaja Saponaria Molina (STIMULON™ (Antigenics, Inc.,Lexington, Mass.) and saponin-based adjuvants, includingimmunostimulating complexes (ISCOMs; structured complex of saponins andlipids) and other ISCOM-based adjuvants, such as ISCOMATRIX™ andAbISCO®-100 and -300 series adjuvants (Isconova AB, Uppsala, Sweden);QS21 and 3 D-MPL together with an oil in water emulsion as disclosed inU.S. Pat. Appl. No. 2006/0073171; stearyl tyrosine (ST) and amideanalogs thereof; virus-like particles (VLPs) and reconstituted influenzavirosomes (IRIVs); complete Freund's adjuvant (CFA); incomplete Freund'sadjuvant (IFA); E. coli heat-labile enterotoxin (LT); immune-adjuvants,including cytokines, such as IL-2, IL-12, GM-CSF, Flt3, accessorymolecules, such as B7.1, and mast cell (MC) activators, such as mastcell activator compound 48/80 (C48/80); water-insoluble inorganic salts;liposomes, including those made from DNPC/Chol and DC Chol; micelles;squalene; squalane; muramyl dipeptides, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S.Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-n-glycero-3-hydroxyphosphoryl;SAF-1 (Syntex); AS05 (GlaxoSmith Kline, Pa.); and combinations thereof.

In preferred embodiments, adjuvant potency may be enhanced by combiningmultiple adjuvants as described above, including combining variousdelivery systems with immunopotentiating substances to formmulti-component adjuvants with the potential to act synergistically toenhance antigen-specific immune responses in vivo. Exemplaryimmunopotentiating substances include the above-described adjuvants,including, for example, MPL and synthetic derivatives, MDP andderivatives, oligonucleotides (CpG etc), ds RNAs, alternativepathogen-associated molecular patterns (PAMPs)(E. coli heat labileenterotoxin; flagellin, saponins (QS-21 etc), small molecule immunepotentiators (SMIPs, e.g., resiquimod [R848]), cytokines, andchemokines.

Pharmaceutically-Acceptable Delivery Vehicles

Pharmaceutically-acceptable delivery vehicles, including those describedabove may be employed to enhance the delivery and/or control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the oligosaccharides,oligosaccharide conjugates, and/or adjuvants. Controlled delivery may beeffected by selecting appropriate macromolecules (for examplepolyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

The oligosaccharide compositions of the present invention, includingoligosaccharide-protein carrier conjugate compositions, can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a pharmaceutically acceptable vehicle (ordiluents). Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences, supra. In order to form apharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe above-described compounds together with a suitable amount of proteincarrier and/or vehicle.

Typically, the immunogenic or immunoprotective compositions may beprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection. An aqueous composition for parenteraladministration, for example, may include a solution of the immunogeniccomponent(s) dissolved or suspended in a pharmaceutically acceptablevehicle or diluent, preferably a primarily aqueous vehicle.Pharmaceutically acceptable vehicles or diluents may include water,saline, including neutral saline solutions buffered with phosphate,Tris, glycerol, ethanol, and the like. An aqueous composition may beformulated as a sterile, pyrogen-free buffered saline orphosphate-containing solution, which may include a preservative or maybe preservative free. Suitable preservatives include benzyl alcohol,parabens, thimerosal, chlorobutanol, and benzalkonium chloride, forexample. Aqueous solutions are preferably approximately isotonic, andits tonicity may be adjusted with agents such as sodium tartrate, sodiumchloride, propylene glycol, and sodium phosphate. Additionally,auxiliary substances required to approximate physiological conditions,including pH adjusting and buffering agents, tonicity adjusting agents,wetting or emulsifying agents, pH buffering substances, and the like,including sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. may be included with the vehicles described herein.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The preparation of such pharmaceutical compositions iswithin the ordinary skill in the art, and may be guided by standardreference books such as Remington's Pharmaceutical Science, supra, whichis incorporated herein by reference.

Compositions may be formulated in a solid or liquid form for oraldelivery. For solid compositions, nontoxic and/or pharmaceuticallyacceptable solid protein carriers may include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. For oral administration, a pharmaceuticallyacceptable nontoxic composition may be formed by incorporating any ofthe normally employed excipients, including those protein carrierspreviously listed, and a unit dosage of an active ingredient, that is,one or more compounds of the invention, whether conjugated to a proteincarrier or not.

Topical application of antibodies to an airway surface, for example, canbe carried out by intranasal administration (e.g., by use of dropper,swab, or inhaler which deposits a pharmaceutical formulationintranasally). Topical application of the antibodies to an airwaysurface can also be carried out by inhalation administration, such as bycreating respirable particles of a pharmaceutical formulation (includingboth solid particles and liquid particles) containing the antibodies asan aerosol suspension, and then causing the subject to inhale therespirable particles. Methods and apparatuses for administeringrespirable particles of pharmaceutical formulations are well known, andany conventional technique can be employed. Oral administration may bein the form of an ingestable liquid or solid formulation.

Further, compositions may be formulated in an aerosol for nasaladministration. For aerosol administration, the immunogenic compoundsare preferably supplied in finely divided form along with one or moresurfactant(s) and/or propellant(s). The surfactant will be nontoxic, andpreferably soluble in the propellant. Representative of such agents arethe esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. The surfactant may constitute0.1%-20% by weight of the composition, preferably 0.25-5%. The balanceof the composition is ordinarily propellant. A protein carrier can alsobe included, as desired, as with, e.g., lecithin for intranasaldelivery.

The concentration of the immunogenic oligosaccharides of the inventionin the pharmaceutical formulations can vary widely, i.e., from less thanabout 0.1%, usually at or at least about 0.1% to as much as 20% to 50%or more by weight, and will be selected primarily by fluid volumes,viscosities, etc., and in accordance with the particular mode ofadministration selected. A human unit dose form of the compounds andcomposition is typically included in a pharmaceutical composition thatcomprises a human unit dose of an acceptable protein carrier, preferablyan aqueous protein carrier, and is administered in a volume of fluidthat is known by those of skill in the art to be used for administrationof such compositions to humans, and is adjusted according to commonlyunderstood principles for a particular subject to be treated. Thus inone embodiment, the invention provides a unit dosage of the vaccinecomponents of the invention in a suitable amount of an aqueous solution,such as 0.1-3 ml, preferably 0.2-2 mL.

Methods of Treatment

The immunogenic and immunoprotective compositions of the presentinvention may be administered to any animal species at risk fordeveloping an infection by M. catarrhalis.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

The amounts effective for inducing an immune response or providingprotective immunity will depend on a variety of factors, including theoligosaccharide composition, conjugation to a protein carrier, inclusionand nature of adjuvant(s), the manner of administration, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician. By way of example, the amounts may generallyrange for the initial immunization (that is for a prophylacticadministration) from about 1.0 μg to about 5,000 μg of carbohydrateantigen for a 70 kg patient, (e.g., 1.0 μg, 2.0 μg, 2.5 μg, 3.0 μg, 3.5μg, 4.0 μg, 4.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg, 17.5 μg, 20μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 250 μg, 500μg, 750 μg, 1,000 μg, 1,500 μg, 2,000 μg, 2,500 μg, 3,000 μg, 3,500 μg,4,000 μg, 4,500 μg or 5,000 μg). The actual dose administered to asubject is often determined according to an appropriate amount per kg ofthe subject's body weight. For example, an effective amount may be about0.1 μg to 5 μg/kg body weight.

A primary dose may optionally be followed by boosting dosages of fromabout 1.0 to about 1,000 of carbohydrate antigen (e.g., 1.0 μg, 2.0 μg,2.5 μg, 3.0 μg, 3.5 μg, 4.0 μg, 4.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg,15 μg, 17.5 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg,100 μg, 250 μg, 500 μg, 750 μg, 1,000 μg, 1,500 μg, 2,000 μg, 2,500 μg,3,000 μg, 3,500 μg, 4,000 μg, 4,500 μg or 5,000 μg) pursuant to aboosting regimen over weeks to months depending upon the patient'sresponse and condition by measuring specific T cell activity in thepatient's blood.

The present invention contemplates the use of single- and multi-valentglycoconjugate vaccines comprising any of the synthetic oligosaccharidesdescribed herein. The identification of a single oligosaccharide antigeneliciting a cross-reactive immune response can facilitate development ofa single-antigen vaccine candidate active against all common M.catarrhalis bacterial serotypes and/or strains.

The present invention further contemplates multi-antigen glycoconjugatevaccines comprising a plurality of any of the synthetic oligosaccharidesdescribed herein so as to provide protection against a single serotypeor serotype subtype of M. catarrhalis or against a plurality ofserotypes or serotype subtypes of M. catarrhalis. Thus, in oneembodiment, for example, the invention provides a composition containingtwo, three, four or more different oligosaccharide antigens according toFormula 1b.

The immunogenic compositions comprising a compound of the invention maybe suitable for use in adult humans or in children, including youngchildren or others at risk for contracting an infection caused by aLOS-expressing bacterial species. Optionally such a composition may beadministered in combination with other pharmaceutically activesubstances, and frequently it will be administered in combination withother vaccines as part of a childhood vaccination program.

Compositions for administration may beneficially include multipleoligosaccharide- or oligosaccharide conjugates that elicit an immuneresponse to a plurality of different epitopes so as to provide increasedprotection against a single strain or serotype of M. catarrhalis oragainst a plurality of strains or serotypes of M. catarrhalis. Moreover,compositions may be administered whereby a prime immunization with oneor multiple antigen conjugates is followed by boosting events with oneor more cross-reactive core conjugates according to the presentinvention.

Antibody Compositions

In another embodiment, the invention provides diagnostic antibodies, aswell as pharmaceutical compositions comprising one or more anti-LOSantibody(ies) or a functional fragment(s) thereof, and a physiologicallyacceptable vehicle. Methods for generating these antibodies are furtherdescribed below.

Pharmaceutical antibody compositions may be used in a method forproviding passive immunity against M. catarrhalis infections. Apharmaceutical antibody composition may be administered to an animalsubject, preferably a human, in an amount sufficient to prevent orattenuate the severity, extent of duration of the infection by one ormore strains or serotypes of M. catarrhalis.

The administration of one or more antibodies may be either prophylactic(prior to anticipated exposure to a bacterial infection) or therapeutic(after the initiation of the infection, at or shortly after the onset ofthe symptoms). The dosage of the one or more antibodies will varydepending upon factors as the subject's age, weight and species. Ingeneral, the dosage of the antibody may be in a range from about 1-10mg/kg body weight. In a preferred embodiment, the antibody is ahumanized antibody of the IgG or the IgA class. The route ofadministration of the one or more antibodies may be oral or systemic,for example, subcutaneous, intramuscular or intravenous.

The use of antibodies as diagnostic agents is further described belowand in U.S. Pat. No. 7,595,307 and U.S. Pat. Appl. Publ. No.2009/0155299, the disclosures of which are incorporated by referenceherein.

The present invention also provides one or more kits useful fordiagnosing, treating, and/or preventing an M. catarrhalis infection. Forexample, the kits may include one or more containers holding thediagnostic or pharmaceutical compositions of the invention. The kits mayalso include other container(s) containing, for example, one or moresolutions necessary or convenient for the particular diagnostic orpharmaceutical use. The container means can be made of glass, plastic orfoil and can be a vial, bottle, pouch, tube, bag, etc. The kit may alsocontain written information, such as procedures for carrying out thepresent invention or analytical information, such as the amount ofreagent contained in the container(s).

Generation of Antibodies and their Use in Assay Development

In a further aspect, the present invention provides compositions andmethods for inducing production of antibodies for use in assaydevelopment, including their use as detection agents and serum screeningtools.

Antisera to LOS-conjugates may be generated in New Zealand white rabbitsby 3-4 subcutaneous injections over 13 weeks. A pre-immune bleed maygenerate about 5 mL of baseline serum from each rabbit. A primeinjection (10 μg antigen equivalent) may be administered as an emulsionin complete Freund's adjuvant (CFA). Subsequent injections (5 μg antigenequivalent) may be given at three week intervals in incomplete Freund'sadjuvant (IFA). Rabbits may be bled every two weeks commencing one weekafter the third immunization. Approximately 25-30 mL of serum per rabbitmay be generated from each bleeding event and frozen at −80° C. Serummay be analyzed by ELISA against the corresponding LOS-conjugate asdescribed below. In addition, antisera from later bleeds may be affinitypurified as further described below.

The oligosaccharides and antibodies of the present invention can be usedas diagnostic reagents for detecting M. catarrhalis LOS antigens orantibodies thereagainst, which are present in biological samples. Thedetection reagents may be used in a variety of immunodiagnostictechniques, known to those of skill in the art, including ELISA- andmicroarray-related technologies. In addition, these reagents may be usedto evaluate antibody responses, including serum antibody levels, toimmunogenic oligosaccharide conjugates. The assay methodologies of theinvention typically involve the use of labels such as fluorescent,chemiluminescent, radioactive, enzymatic labels or dye molecules, and/orsecondary immunologic reagents for direct or indirect detection of acomplex between an antigen or antibody in a biological sample and acorresponding antibody or antigen bound to a solid support.

Such assays typically involve separation of unbound antibody in a liquidphase from a solid phase support to which antibody-antigen complexes arebound. Solid supports which can be used in the practice of the inventioninclude substrates such as nitrocellulose (e.g., in membrane ormicrotiter well form); polyvinylchloride (e.g., sheets or microtiterwells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like.

Typically, a solid support is first reacted with a first bindingcomponent (e.g., an antigen or antibody in accordance with the presentinvention) under suitable binding conditions such that the first bindingcomponent is sufficiently immobilized to the support. In some cases,mobilization to the support can be enhanced by first coupling theantibody or oligosaccharide to a protein with better binding properties,or that provides for immobilization of the antibody or antigen on thesupport without significant loss of antibody binding activity orspecificity. Suitable coupling proteins include, but are not limited to,macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind antibodies thesupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like. Suchmolecules and methods of coupling these molecules are well known tothose of ordinary skill in the art and are described in, e.g., U.S. Pat.No. 7,595,307 and U.S. Pat. Appl. No. US 2009/0155299.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Example 1 Synthesis of Monosaccharide Building Blocks Synthesis of allyl4,6-O-benzylidene-2-O-pivaloyl-α-D-glucopyranoside 1 (FIG. 4A)

Allyl alcohol (500 mL, 7.35 mol) was cooled to 0° C. and acetyl chloride(72 mL, 1.0 mol) was added slowly, keeping the reaction mixture below 5°C. D-Glucose monohydrate (200 g, 1.0 mol) was added and the reactionmixture was stirred overnight at room temperature, then overnight at 40°C. The solvent was evaporated to provide a thick syrup, which wasco-evaporated with toluene (500 mL) and used without furtherpurification.

Crude allyl-D-glucopyranoside was dissolved in acetonitrile (1000 mL).To this stirred solution were added benzaldehyde dimethylacetal (250 mL,1.7 mol) and (+/−)-camphor-10-sulfonic acid (12 g, 0.05 mol) and thesolution was stirred at room temperature overnight. The solids werefiltered off, the solvent was removed in vacuo and the residue was takenup in ethyl acetate (1000 mL), washed with saturated NaHCO₃ (3×200 mL)and water (3×200 mL) then evaporated to a thick syrup. The syrup wasdissolved in hot ethanol (500 mL) and cooled to −20° C. for 2.5 days.The solid product was filtered and washed with cold ethanol, and driedunder vacuum to give allyl-4,6-O-benzylidene-α-D-glucopyranoside (150 g,48%).

Allyl-4,6-O-benzylidene-α-D-glucopyranoside (51 g, 165 mmol) wasdissolved in pyridine (200 mL) and cooled to −20° C. Pivaloyl chloride(22 mL, 180 mmol) was added dropwise to the stirred solution over 20minutes. After 1 hour at −20° C. another aliquot of pivaloyl chloride(22 mL, 180 mmol) was added over 20 minutes. After another 1 hour at−20° C. a final aliquot of pivaloyl chloride (22 mL, 180 mmol) was addedover 20 minutes and stirring continued at −20° C. for an additional 1hour. The reaction was quenched with methanol (40 mL) and allowed towarm to room temperature. The reaction mixture was diluted with ethylacetate (600 mL) and washed with water (2×400 mL), brine (300 mL), 1 mNaOH (2×300 mL) and brine (300 mL), dried over Na₂SO₄, filtered andconcentrated to give allyl4,6-O-benzylidene-2-O-pivaloyl-α-D-glucopyranoside 1 as a crude productas a yellow oil (60 g, 92%).

Synthesis of 2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranosyltrichloroacetimidate 2 (FIG. 4B)

3,4,6-Tri-O-acetyl-D-glucal (5.0 g, 18.4 mmol) was dissolved in methanol(100 mL) and a solution of sodium methoxide in methanol (0.25 mL, 25%solution by weight, 1.1 mmol). The solution was stirred at roomtemperature for 3 hours. The solvent was removed and the resultingresidue was co-evaporated with toluene (3×10 mL) before being taken upin a 4:1 solution of NMP:THF and cooled to 0° C. Solid NaH (3.3 g of 60%suspension, 82.5 mmol) was added and the mixture was stirred for 30minutes before the addition of BnBr. The reaction mixture was stirred atroom temperature overnight, quenched with methanol and the solventremoved. The residue was suspended in ethyl acetate, washed with 1 NHCl, brine, and saturated NaHCO₃ (100 mL each). The ethyl acetatesolution was dried over MgSO₄, filtered and concentrated in vacuo. Thecrude material was purified on a silica gel column (80 g) using an ISCOautomated chromatography system, eluting with a 0→50% gradient of ethylacetate in heptane, to give 3,4,6-tri-O-benzyl-D-glucal (7.2 g, 94%).

3,4,6-tri-O-benzyl-D-glucal (7.2 g, 17.3 mmol) in CH₂Cl₂/acetone (2:1,105 mL) and a saturated solution of NaHCO₃ (100 mL) was added. Themixture was stirred vigorously while a solution of Oxone (21.2 g, 34.6mmol) in water (150 mL) was added dropwise over 30 minutes. After 1.5hours of vigorous stirring the layers were separated, and the aqueouslayer was extracted with CH₂Cl₂ (2×150 mL). The organic solutions werecombined, dried over MgSO₄, filtered and concentrated. The residue wasco-evaporated with toluene (3×10 mL), dissolved in allyl alcohol (25 mL)and stirred at room temperature overnight. The reaction mixture waspurified on a silica gel column (80 g) using an ISCO automatedchromatography system, eluting with a 0→60% gradient of ethyl acetate inheptane, to give allyl 3,4,6-tri-O-benzyl-β-D-glucopyranoside (4.2 g,50%).

Allyl 3,4,6-tri-O-benzyl-β-D-glucopyranoside (4.2 g, 8.6 mmol) wasdissolved in CH₂Cl₂ (100 mL) and pivaloyl chloride (1.7 mL, 12.9 mmol)and DMAP (1.6 g, 13.1 mmol) were added. The reaction mixture was stirredat room temperature for 18 hours, quenched with methanol, diluted withCH₂Cl₂, washed with saturated NH₄Cl solution, brine, saturated NaHCO₃(100 mL each), dried over MgSO₄, filtered and concentrated to give crudeallyl 2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranoside (4.7 g, 95%).

Allyl 2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranoside (3.2 g, 5.6mmol) was dissolved in dry THF (20 mL) under an inert atmosphere. In aseparate flask 1,5-cyclooctadienebis(methyldiphenylphosphine)-iridium(I)hexafluorophosphate (100 mg, 0.12 mmol) was suspended in dry THF (10 mL)under an inert atmosphere. H₂ gas was bubbled through the catalystsolution for 10 minutes during which the red suspension turned into aclear pale yellow solution. The solution was purged by N₂ gas bubblingfor 10 minutes. The catalyst solution was then added to the allylglycoside solution and stirred for 15 minutes. Next NMO (50% aqueous, 5mL) and OsO₄ (25 mg, XX mmol) were added and the dark mixture wasstirred for 2 hours. The reaction mixture was diluted with ethylacetate, washed twice with brine and saturated NaHCO₃ (100 mL each),dried over Na₂SO₄, filtered and concentrated. The crude material waspurified on a silica gel column (40 g) using an ISCO automatedchromatography system, eluting with a 0→100% gradient of ethyl acetatein heptane, to give 2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranose(2.65 g, 89%).

2-Pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranose (8.2 g, 15.3 mmol) wasdissolved in CH₂Cl₂ (50 mL) and trichloroacetonitrile (15 mL) was addedfollowed by K₂CO₃ (10 g, 72 mmol). The mixture was stirred at roomtemperature for 8 hours, filtered and concentrated. The residue waspassed through a plug of silica gel (washed previously with 1% Et₃N inheptanes) by elution with 3:1 heptane: ethyl acetate. Removal of thesolvent gave the desired trichloroacetimidate product,2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranosyl trichloroacetimidate 2(10.17 g, 97%).

Example 2 Synthesis of Tetramer Core and Conjugates Thereof Synthesis ofTetramer Core 5 (FIG. 5A)

As shown in FIG. 5A, allyl4,6-O-benzylidene-2-O-pivaloyl-α-D-glucopyranoside 1 (2.0 g, 5.1 mmol)and 2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranosyltrichloroacetimidate 2 (4.1 g, 6.1 mmol) were combined, co-evaporatedwith toluene (3×10 mL) and dissolved in dry CH₂Cl₂ (50 mL). Freshlyactivated AW-300 molecular sieves (3 g) were added and the reactionmixture was stirred for 10 minutes and cooled to 0° C. A solution oftrimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.09 mL, 0.5 mmol) inCH₂Cl₂ (0.9 mL) was added and the reaction stirred for 30 minutes. Thereaction mixture was filtered, quenched with Et₃N (1 mL) and the solventremoved to give the crude coupling product, which was used withoutfurther purification.

The crude coupling product was dissolved in methanol: tetrahydrofuran(1:1, 100 mL) and sodium methoxide solution (10 mL, 25% by weight, 44mmol) was added. The solution was stirred at room temperature for 18hours, then diluted with ethyl acetate (200 mL), washed with 1N HCl (100mL), brine (100 mL), and saturated NaHCO₃ (100 mL), dried over MgSO₄,filtered and concentrated. The crude material was purified on a silicagel column (300 mL), eluting with 40:60 ethyl acetate:heptane, to givethe desired disaccharide diol 57 (2.63 g, 70%).

The disaccharide diol 57 (2.63 g, 3.55 mmol) was dissolved in dry DMF(40 mL) and cooled to 0° C. Solid NaH (0.57 g of 60% suspension, 14.2mmol) was added and the mixture was stirred for 10 minutes before theaddition of BnBr (1.25 mL, 10.6 mmol). The reaction mixture was stirredat room temperature for 18 hours and quenched with methanol. Thesolution was diluted with ethyl acetate, washed with 1 N HCl, brine, andsaturated NaHCO₃ (100 mL each). The ethyl acetate solution was driedover MgSO₄, filtered and concentrated in vacuo to give crude product,which was used without further purification.

The crude benzylation product was taken up in 80% acetic acid (50 mL)and heated at 50° C. for 4 hours. The reaction volume was reduced by 70%under vacuum then diluted with ethyl acetate (100 mL) and washed withwater (3×100 mL) and saturated NaHCO₃ (2×100 mL), dried over MgSO₄,filtered and concentrated. The crude material was purified on a silicagel column (80 g) using an ISCO automated chromatography system, elutingwith a 0→100% gradient of ethyl acetate in heptane, to give allyl2-O-benzyl-3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-α-D-glucopyranoside3 (2.07 g, 70%).

The trichloroacetimidate donor 2 (257 mg, 0.378 mmol) and the diolacceptor 3 (105 mg, 0.126 mmol) were combined and co-evaporated withtoluene (2×5 mL) then dissolve in dry CH₂Cl₂ (3 mL). Freshly activatedAW-300 molecular sieves (0.5 g) were added and the reaction mixture wasstirred for 10 minutes. A solution of trimethylsilyltrifluoromethanesulfonate (TMSOTf, 4.9 μL, 0.027 mmol) in CH₂Cl₂ (45 μL)was added and the reaction stirred for 30 minutes. The reaction mixturewas quenched with Et₃N (0.25 mL), filtered and the solvent removed togive the crude coupling product, which was purified on a silica gelcolumn (40 g) using an ISCO automated chromatography system, elutingwith a 0→70% gradient of ethyl acetate in heptane, to give the desiredtetrasaccharide coupling product (45 mg, 19%). Trisaccharide productresulting from single coupling to the 6-OH position of the acceptor wasalso recovered as a by-product.

Tetrasaccharide was dissolved in methanol: tetrahydrofuran (1:1, 2 mL)and sodium methoxide solution (0.2 mL, 25% by weight, 0.88 mmol) wasadded. The solution was stirred at room temperature overnight, thendiluted with ethyl acetate (50 mL), washed with 1N HCl (50 mL), brine(50 mL), and saturated NaHCO₃ (50 mL), dried over MgSO₄, filtered andconcentrated. The crude coupling product was purified on a silica gelcolumn (12 g) using an ISCO automated chromatography system, elutingwith a 0→70% gradient of ethyl acetate in heptane, to give the desiredtetrasaccharide product 4 (25 mg, 61%).

Tetrasaccharide 4 (375 mg, 0.221 mmol) was dissolved in dioxane (6 mL)under N₂ and AlBN (20 mg, 0.122 mmol) and thioacetic acid (0.50 mL, 7.1mmol) were added. The reaction mixture was heated to 75° C. under aninert atmosphere for 2 hours, at which point another aliquot of AlBN (50mg, 0.30 mmol) was added and stirring continued for 2 hours. Thereaction was quenched by the addition of cyclohexene (1 mL) and after 15minutes at 75° C. the reaction was cooled and the solvent was removed.The crude product was purified on a silica gel column (40 g) using anISCO automated chromatography system, eluting with a 0→75% gradient ofethyl acetate in heptane, to give the desired tetrasaccharide thiolproduct (310 mg, 79%).

Tetrasaccharide thiol (310 mg, 0.175 mmol) was coevaporated with toluene(3×10 mL) and dissolved in dry THF (5 mL). In a jacketed flask equippedwith a dry ice condenser and cooled to −78° C., NH₃ gas was condensedinto the flask under an Ar atmosphere until the liquid volume reachedapproximately 20 mL. The tetrasaccharide thiol solution was transferredby cannula into the liquid NH₃. Sodium metal (250 mg, 10.9 mmol) wasadded under positive Ar gas flow and once a permanent blue color wasestablished the reaction was stirred for another 20 minutes. Thereaction was quenched at −78° C. by the addition of saturated NH₄Clsolution (10 mL) and warmed to room temperature. N₂ was bubbled throughthe reaction mixture to remove excess NH₃, and then the solution wasconcentrated to 2 mL. The crude material was purified by size exclusionchromatography on a bed of BioGel P4 by elution with water. Thefractions containing product were concentrated to yield the desiredMoraxella tetrasaccharide core 5 (75 mg, 58%).

Synthesis of Tetramer Core Conjugates 49, 50 (FIG. 5B)

A solution of tris(2-carboxyethyl)phosphine (TCEP) in water (8.6 μL,0.05 M, 0.43 μmol) was added to a solution of tetrasaccharide thiol 5 inwater (8.3 mg/mL, 72 μL, 0.86 μmol), and the resulting solution wasstirred for 1 hour. A solution of maleimide-activated keyhole limpethemocyanin (Imject® KLH, Pierce, Rockford, Ill.) (0.5 mL, 5 mg, ˜0.43μmol maleimide) was added and the resulting solution stirred overnightat room temperature. The reaction mixture was purified by de-salting onD-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). The column waspre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.77159 which contains sodium chloride, sodium phosphate monobasic, sodiumphosphate dibasic), the crude material loaded onto the column and elutedwith purification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired tetramer-KLHconjugate 49 (4.61 mg).

A solution of tris(2-carboxyethyl)phosphine (TCEP) in water (30 μL, 0.05M, 1.5 μmol) was added to a solution of tetrasaccharide thiol 5 in water(8.3 mg/mL, 265 μL, 3.0 μmol), and the resulting solution was stirredfor 1 hour. A solution of maleimide-activated bovine serum albumin(Imject® BSA, Pierce, Rockford, Ill.) (0.5 mL, 5 mg, ˜1.5 μmolmaleimide) was added and the resulting solution stirred overnight atroom temperature. The reaction mixture was purified by de-salting onD-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). The column waspre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.77159), the crude material loaded onto the column and eluted withpurification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired tetramer-BSAconjugate 50 (4.07 mg).

Example 3 Synthesis of Heptamer Core and Conjugates Thereof Synthesis ofHeptamer Core 46 (FIG. 6A)

As shown in FIG. 6A, allyl2-O-benzyl-3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-4-O-(3,4,6-tri-O-benzyl-β-D-glucopyanosyl)-6-O-(4-methoxyphenyl)-α-D-glucopyranoside15 (2.09 g, 1.52 mmol; the synthesis which is described in Example 7below) was dissolved in a 4:1 solution of THF:NMP (15 mL) and cooled to0° C. Solid NaH (305 mg of 60% suspension, 7.62 mmol) was added and themixture was stirred for 10 minutes before the addition of benzyl bromide(300 μL, 6.09 mmol). The reaction mixture was stirred at roomtemperature for 18 hours, quenched with methanol, stirred for 30minutes, diluted with ethyl acetate (100 mL), and washed with water andbrine (100 mL each). The ethyl acetate solution was dried over Na₂SO₄,filtered and concentrated in vacuo. The crude material was purified bysilica gel chromatography using toluene/ethyl acetate as the eluent togive the desired trisaccharide (1.62 g, 73%).

The benzylated trisaccharide (721 mg, 0.493 mmol) was taken up inacetonitrile and water (95:5, 18.9 mL) and cooled to 0° C. Ammoniumcerium(IV) nitrate (CAN, 811 mg, 1.48 mmol) was added and the reactionmixture was stirred for 4 hours. Ethyl acetate (50 mL) was added and themixture was washed twice with water, then with brine, dried over Na₂SO₄,filtered and concentrated. The crude residue was purified by silica gelchromatography eluting with ethyl acetate in heptane to give the desiredtrisaccharide alcohol 43 (568 mg, 85%).

Allyl2-O-benzyl-3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-4-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyanosyl)-α-D-glucopyranoside43 (502 mg, 0.370 mmol) and2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranosyl trichloroacetimidate 2(501 mg, 0.740 mmol) were dissolved in dry CH₂Cl₂ (5.5 mL). Freshlyactivated AW-300 molecular sieves (0.5 g) were added and the reactionmixture was stirred for 30 minutes and cooled to 0° C. Trimethylsilyltrifluoromethanesulfonate (TMSOTf, 7 μL, 0.0.038 mmol) was added and thereaction stirred for 40 minutes. The reaction mixture was quenched withEt₃N (52 μL), and diluted with CH₂Cl₂, filtered through celite and thesolvent removed. The crude residue was purified by silica gelchromatography, eluting with heptane: ethyl acetate to give the desiredtetrasaccharide coupling product (620 mg, 90%).

The tetrasaccharide coupling product (620 mg, 0.33 mmol) was dissolvedin methanol: tetrahydrofuran (1:1, 10 mL) and sodium methoxide solution(1.0 mL, 25% by weight, 4.4 mmol) was added. The solution was stirred atroom temperature for 18 hours, then heated to 45° C. for 6 hours,quenched with 1N HCl, diluted with ethyl acetate (100 mL), washed withbrine (100 mL), and saturated NaHCO₃ (100 mL), dried over Na₂SO₄,filtered and concentrated. The crude material was purified by silica gelchromatography using ethyl acetate in heptane, to give the desiredtetrasaccharide alcohol 44 (447 mg, 67%).

Thiomethyl4-O-(2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-β-D-glucopyranoside16 (750 mg, 0.522 mmol) (J. Org. Chem., 1996, 61:7711) was dissolved indry CH₂Cl₂ (8 mL) under nitrogen and cooled to 0° C. Bromine (66 μL,1.29 mmol) was added and the reaction mixture stirred for 20 minutes.The reaction was quenched with saturated Na₂S₂O₃ solution (50 mL), andthe aqueous layer was extracted with CH₂Cl₂ (3×50 mL). The combinedorganic extracts were washed with saturated NaHCO₃ and brine, dried overNa₂SO₄, filtered and concentrated to give the crude glycosyl bromide,which was used without further purification.

The glycosyl bromide and the tetrsaccharide acceptor 44 (825 mg, 0.60mmol) were combined, 4 Å molecular sieves (700 mg) were added, and themixture taken up in anhydrous diethyl ether (8 mL) under nitrogen andcooled to −40° C. After stirring for 30 minutes, silver triflate (140mg, 0.547 mmol) was added and the reaction mixture was stirred for 1hour. The reaction was quenched at −20° C. by pouring into saturatedsodium ascorbate solution (50 mL) and stirring for 30 minutes. Themixture was filtered through celite and the filtrate washed with ethylacetate (3×50 mL). The organic layer was washed with saturated NaHCO₃,brine, dried over Na₂SO₄, filtered and concentrated. The crude residuewas purified by silica gel chromatography using a gradient of ethylacetate in toluene, to give the desired heptasaccharide 45 (488 mg,89%).

Heptasaccharide 45 (475 mg, 0.150 mmol) was dissolved in dioxane (6 mL),thioacetic acid (0.5 mL, 7.1 mmol) was added, and the solution purgedwith argon gas for 10 minutes. The solution was purged with nitrogen gasfor 15 minutes before AlBN (50 mg, 0.30 mmol) was added and the reactionmixture heated to 85° C. under an inert atmosphere overnight. Thesolvent was removed and the crude product was co-evaporated with toluene(2×10 mL). The crude residue was purified by size exclusionchromatography using BioRad SX-1 Bio-Beads (2.5 cm×25 cm column) andtoluene as the eluent. Appropriate fractions were combined to give thedesired octasaccharide thioacetate product (400 mg, 82%).

Heptasaccharide thioacetate (400 mg, 0.123 mmol) was co-evaporated withtoluene (2×10 mL) and dissolved in dry THF (10 mL). In a flame-driedjacketed flask equipped with a dry ice condenser and cooled to −78° C.,NH₃ gas was condensed into the flask under an Ar atmosphere until theliquid volume reached approximately 30 mL. The heptasaccharidethioacetate solution was transferred by cannula into the liquid NH₃.Sodium metal (250 mg, 10.9 mmol) was added under positive Ar gas flowand once a permanent blue color was established the reaction was stirredfor another 20 minutes. The reaction was quenched at −78° C. by theaddition of saturated NH₄Cl solution (10 mL) and warmed to roomtemperature. N₂ was bubbled through the reaction mixture to removeexcess NH₃, and then the solution was concentrated to dryness. The crudematerial was purified by size exclusion chromatography on a bed ofBioGel P4 (2.5 cm×60 cm column) by elution with water. The fractionscontaining product were concentrated to yield the desired Moraxellaheptasaccharide core 46 (45 mg, 30%).

Synthesis of Heptamer Core Conjugates 47, 48 (FIG. 6B)

A solution of tris(2-carboxyethyl)phosphine (TCEP) in water (8.6 μL,0.05 M, 0.43 μmol) was added to a solution of heptasaccharide thiol 46in water (12.6 mg/mL, 87 μL, 0.86 μmol), and the resulting solution wasstirred for 1 hour. A solution of maleimide-activated keyhole limpethemocyanin (Imject® KLH, Pierce, Rockford, Ill.) (0.5 mL, 5 mg, ˜0.43μmol maleimide) was added and the resulting solution stirred overnightat room temperature. The reaction mixture was purified by de-salting onD-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). The column waspre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.77159), the crude material loaded onto the column and eluted withpurification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desiredheptasaccharide core-KLH conjugate 47.

A solution of tris(2-carboxyethyl)phosphine (TCEP) in water (30 μL, 0.05M, 1.5 μmol) was added to a solution of heptasaccharide thiol 46 inwater (12.6 mg/mL, 294 μL, 3.0 μmol), and the resulting solution wasstirred for 1 hour. A solution of maleimide-activated bovine serumalbumin (Imject® BSA, Pierce, 5 mg, 1.5 μmol maleimide) in Imject®Conjugation Buffer (Pierce, 250 μL diluted with water, 250 μL) was addedand the resulting solution stirred overnight at room temperature. Thereaction mixture was purified by de-salting on D-Salt P-6000 10 mLcolumn (Pierce, Rockford, Ill.). The column was pre-equilibrated with 30mL of purification buffer (Pierce, Prod. No. 77159), the crude materialloaded onto the column and eluted with purification buffer. 1-mLfractions were collected and analyzed for protein content by absorbanceat 280 nm (A₂₈₀). Fractions containing protein were combined andlyophilized to give the desired heptasaccharide core-BSA conjugate 48.

Example 4 Synthesis of Serotype A Octamer and Conjugates ThereofSynthesis of Serotype A Octamer 27 (FIG. 7A)

Allyl2-O-benzyl-3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-4-O-(3,4,6-tri-O-benzyl-β-D-glucopyranosyl)-6-O-(4-methoxyphenyl)-α-D-glucopyranoside15 (680 mg, 0.50 mmol) and2-deoxy-2-azido-3-O-benzyl-4,6-di-O-acetyl-D-glucopyranosyltrichloroacetimidate (525 mg, 1.0 mmol) were combined, co-evaporatedwith toluene (2×10 mL) and dissolved in dry ether (10 mL). The synthesisof 15 is further described in Example 7 below. Freshly activated AW-300molecular sieves (1 g) were added and the reaction mixture was stirredfor 10 minutes and cooled to 0° C. A solution of trimethylsilyltrifluoromethanesulfonate (TMSOTf, 9 μL, 0.05 mmol) in CH₂Cl₂ (0.08 mL)was added and the reaction stirred for 45 minutes, at which point thereaction mixture was quenched with Et₃N (1 mL), filtered and the solventremoved. The crude coupling product was used without furtherpurification.

The crude tetrasaccharide coupling product was dissolved inmethanol:tetrahydrofuran (1:1, 10 mL) and sodium methoxide solution (1.0mL, 25% by weight, 4.4 mmol) was added. The solution was stirred at roomtemperature for 18 hours, diluted with ethyl acetate (100 mL), quenchedwith 1N HCl, washed with brine (100 mL), and saturated NaHCO₃ (100 mL),dried over MgSO₄, filtered and concentrated. The crude material waspurified on a silica gel column (80 g) using an ISCO automatedchromatography system, eluting with a 0→40% gradient of ethyl acetate intoluene, to give the desired tetrasaccharide diol 23 (470 mg, 57%) plus,separately, the corresponding β-anomer (100 mg, 12%).

Tetrasaccharide diol 23 (635 mg, 0.385 mmol) was co-evaporated withtoluene (2×5 mL) before being taken up in a 4:1 solution of NMP:THF (10mL) and cooled to 0° C. Solid NaH (46 mg of 60% suspension, 1.16 mmol)was added and the mixture was stirred for 10 minutes before the benzylbromide (114 μL, 0.96 mmol) was added. The reaction mixture was stirredat room temperature for 18 hours, quenched with methanol, diluted withethyl acetate (50 mL), washed with 1 N HCl, brine, and saturated NaHCO₃(50 mL each). The ethyl acetate solution was dried over Na₂SO₄, filteredand concentrated in vacuo. The crude material was purified on a silicagel column (40 g) using an ISCO automated chromatography system, elutingwith a 0→60% gradient of ethyl acetate in heptane, to give the desiredtetrasaccharide (600 mg, 85%).

The tetrasaccharide (600 mg, 0.328 mmol) was taken up in acetonitrileand water (4:1, 15 mL) and cooled to 0° C. Ammonium cerium(IV) nitrate(CAN, 540 mg, 0.984 mmol) was added and the reaction mixture was stirredfor 2 hours. The reaction was quenched with Et₃N (2 mL), filteredthrough a small pad of celite, and washed twice with CH₂Cl₂ (50 mL). Theorganics were combined, dried over Na₂SO₄, filtered and concentrated.The crude material was purified on a silica gel column (40 g) using anISCO automated chromatography system, eluting with a 0→75% gradient ofethyl acetate in heptane, to give the desired tetrasaccharide alcohol 24(410 mg, 73%).

Tetrasaccharide acceptor 24 (410 mg, 0.24 mmol) and di-n-butylphosphoryl2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranoside X (210 mg, 0.29 mmol)were combined, co-evaporated with toluene (3×5 mL), dissolved in dryCH₂Cl₂ (5 mL) and cooled under nitrogen to −30° C. The reaction mixturewas stirred for 10 minutes before trimethylsilyltrifluoromethanesulfonate (TMSOTf, 53 μL, 0.29 mmol) was added. Thereaction mixture was stirred for 30 minutes then quenched with Et₃N (2mL), and the solvent removed to give the crude coupling product, whichwas used without further purification.

The crude coupling product was dissolved in methanol: tetrahydrofuran(2:1, 15 mL) and sodium methoxide solution (5.0 mL, 25% by weight, 22mmol) was added. The solution was stirred at room temperature for 18hours and concentrated in vacuo. The residue was partitioned betweenethyl acetate (150 mL) and saturated NH₄Cl (100 mL), washed with brine(2×100 mL), dried over MgSO₄, filtered and concentrated. The crudematerial was purified on a silica gel column (80 g) using an ISCOautomated chromatography system, eluting with a 0→60% gradient of ethylacetate in heptane, to give the desired pentasaccharide alcohol 25 (375mg, 73%).

Thiomethyl4-O-(2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-β-D-glucopyranoside16 (800 mg, 0.557 mmol) (J. Org. Chem. 61 (1996) 7711) was co-evaporatedwith toluene (3×5 mL) then dissolved in dry CH₂Cl₂ (5 mL) under nitrogenand cooled to 0° C. Bromine (30 μL, 0.585 mmol) was added and thereaction mixture stirred for 10 minutes. Dry toluene (10 mL) was addedand removed in vacuo. The toluene azeotrope was repeated three times togive the crude glycosyl bromide, which was used without furtherpurification.

The glycosyl bromide and the pentasaccharide acceptor 25 (336 mg, 0.156mmol) were combined and co-evaporated with toluene (3×5 mL). Freshlyactivated 4 Å molecular sieves (1 g, Linde 600 mesh) were added followedby anhydrous diethyl ether (10 mL) under nitrogen. The mixture wascooled to −40° C. and after 30 minutes silver triflate (143 mg, 0.557mmol) was added and the reaction mixture was stirred for 30 minutes. Thereaction was quenched at −20° C. with Et₃N (0.4 mL), diluted with CH₂Cl₂(50 mL) and filtered through celite and the filtrate washed with CH₂Cl₂(50 mL). The filtrate was stirred with saturated sodium ascorbatesolution for 1 hour then filtered through celite. The filtrate waswashed with saturated NaHCO₃ (100 mL), brine (100 mL), dried overNa₂SO₄, filtered and concentrated. The crude residue was purified bysilica gel chromatography using toluene: ethyl acetate as the eluent togive the desired Serotype A octasaccharide 26 (335 mg, 60%).

Octasaccharide 26 (280 mg, 0.079 mmol) was dissolved in dioxane (10 mL),thioacetic acid (1.0 mL, 14 mmol) and AlBN (60 mg, 0.36 mmol) wereadded, and the solution purged with nitrogen gas for 15 minutes. Thereaction mixture heated to 75° C. under an inert atmosphere for 18hours, cooled to room temperature and quenched with cyclohexene (0.1mL). The solvent was removed and the crude product was purified by sizeexclusion chromatography using BioRad SX-1 Bio-Beads (2.5 cm×60 cmcolumn) and toluene as the eluent to give the desired octaccharidethioacetate (221 mg, 77%).

The octasaccharide thioacetate (26.1 mg, 7.2 μmol) was dissolved in DMF(3 mL) and ethanol (1.5 mL), and the solution was purged with nitrogenfor 5 minutes. Benzyl chloride (30 μL, 0.26 mmol) and 1.0 M NaOH (0.1mL) were added, and the reaction mixture was stirred for 1 hour. Themixture was diluted with ethyl acetate (50 mL), washed with water (2×50mL) and brine (2×50 mL), dried over Na₂SO₄, filtered and concentrated toa yellow oil (26 mg, 98%), which was used without further purification.

Octasaccharide benzyl thioether (26 mg, 7.1 μmol) was dissolved in dryTHF (2 mL). In a flame-dried jacketed flask equipped with a dry icecondenser and cooled to −78° C., NH₃ gas was condensed into the flaskunder an Ar atmosphere until the liquid volume reached approximately 15mL. The octasaccharide benzyl thioether solution was transferred intothe liquid NH₃. Sodium metal (20 mg, 0.87 mmol) was added under positiveAr gas flow and stirred for 10 minutes. The reaction was quenched at−78° C. by the addition of saturated NH₄Cl solution (0.1 mL) and warmedto room temperature. N₂ was bubbled through the reaction mixture toremove excess NH₃, and then the solution was concentrated to dryness.The crude material was purified by size exclusion chromatography on abed of Sephadex G-10 (2.5 cm×8 cm column) by elution with water. Thefractions containing product were concentrated to yield the crudeoctasaccharide (10 mg, quantitative).

The crude octasaccharide (10 mg) was dissolved in water (0.6 mL) andmethanol (0.3 mL), solid NaHCO₃ (20 mg), and acetic anhydride (20 μL)were added. The reaction mixture was stirred for 2 hours then loadeddirectly onto a Sephadex G-10 column (2.5 cm×8 cm) and eluted withwater. Lyophilization of the appropriate fractions gave the desiredMoraxella serotype A octasaccharide 27 (5.2 mg, 50%).

Synthesis of Serotype A Octamer Conjugates 28, 29 (FIG. 7B)

First, a conjugation stock solution of octasaccharide 27 was prepared asfollows. The octasaccharide 27 (5.5 mg, 3.85 μmol) was dissolved inwater (0.5 mL). Hydrazine in water (520 μL, 0.05 M, 60 μL) was added tothe reaction mixture, was stirred at room temperature for 1 hour andconcentrated in vacuo. The residue was co-evaporated with water (3×1 mL)and redissolved in water (300 μL). A solution oftris(2-carboxyethyl)phosphine (TCEP) in water (39 μL, 0.05 M, 1.95 μmol)was added and stirred for 1 hour. Imject® Conjugation Buffer (Pierce,300 μL) was added to provide a stock solution for conjugation to KLH andBSA.

To synthesize the KLH conjugate 28, conjugation stock solution ofoctasaccharide thiol 27 (140 μL, 0.86 μmol) was added to a solution ofmaleimide-activated keyhole limpet hemocyanin (Imject® KLH, Pierce,Rockford, Ill.) (5 mg, 0.43 μmol maleimide) in water (0.5 mL) was addedand the resulting solution stirred overnight at room temperature. Thereaction mixture was purified by de-salting on D-Salt P-6000 10 mLcolumn (Pierce, Rockford, Ill.). The column was pre-equilibrated with 30mL of purification buffer (Pierce, Prod. No. 77159), the crude materialloaded onto the column and eluted with purification buffer. 1-mLfractions were collected and analyzed for protein content by absorbanceat 280 nm (A₂₈₀). Fractions containing protein were combined andlyophilized to give the desired serotype A octamer-KLH conjugate 28.

To synthesize the BSA conjugate 29, conjugation stock solution ofoctasaccharide thiol 37 (500 μL, 3.0 μmol) was added to a solution ofmaleimide-activated bovine serum albumin (Imject® BSA, Pierce, Rockford,Ill.) (5 mg, ˜1.5 μmol maleimide) in Imject® Conjugation Buffer (Pierce,300 μL diluted with water, 300 μL) and the resulting solution stirredfor 18 hours at room temperature. The reaction mixture was purified byde-salting on D-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). Thecolumn was pre-equilibrated with 30 mL of purification buffer (Pierce,Prod. No. 77159), the crude material loaded onto the column and elutedwith purification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired serotype Aoctamer-BSA conjugate 29.

Example 5 Synthesis of Serotype B7 Hexasaccharide Core and ConjugatesThereof Synthesis of Serotype B7 Hexasaccharide Core 8 (FIG. 8A)

In a jacketed 4-L flask D-Glucose-penta-O-acetate (1.0 kg, 2.56 mol) wasdissolved in CH₂Cl₂ (3 L) and p-thiocresol (350.2 g, 2.82 mol) wasadded. The stirred solution was brought to 5° C. and tin(IV) chloride(500 g, 1.9 mol) was added slowly while maintaining an internaltemperature of <10° C. The reaction mixture was stirred overnight at 20°C. The reaction was quenched by transferring the mixture into ice water(4 L). The CH₂Cl₂ layer was washed with saturated NaHCO₃ (2 L), whichwas back-extracted with CH₂Cl₂ (0.5 L). The combined organic layers werewashed with water (3×2 L) then evaporated to a thick syrup. 2-Propanol(4 L) was added and the suspension heated to 70° C. until a clearsolution resulted. The solution was allowed to cool with stirring over 2days. The solids were filtered, washed with 2-propanol and dried invacuo to give p-thiophenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside(595 g, 51%).

p-Thiophenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (100 g, 0.22mol) was dissolved in anhydrous methanol (500 mL) and sodium methoxidein methanol (5 mL, 25% solution by weight, 22 mmol) was added. Thesolution was stirred at room temperature for 2 hours then was quenchedwith A-15 (H⁺) resin (10 g), filtered and concentrated to give thedesired product as a crude syrup (56 g), which was used without furtherpurification

p-Thiophenyl β-D-glucopyranoside (56 g, 0.2 mol) was dissolved inacetonitrile (250 mL). to this stirred solution were added benzaldehydedimethylacetal (60 mL, 0.41 mol) and (+/−)-camphor-10-sulfonic acid (12g, 0.05 mol) and the solution was stirred at room temperature overnight.The solvent was removed and the crude product crystallized from hotethanol to give p-thiophenyl 4,6-O-benzylidene-β-D-glucopyranoside (38g, 52%).

p-Thiophenyl 4,6-O-benzylidene-β-D-glucopyranoside (85 g, 0.23 mol) wasdissolved in dry NMP:THF (3:7, 500 mL) and cooled to 0° C. withstirring. Solid NaH (24 g of 60% suspension, 0.6 mol) was added slowlyand the mixture was stirred for 15 minutes. Benzyl bromide (68 mL, 0.57mol) was added slowly to keep the internal temperature below 20° C. Thereaction mixture was stirred at room temperature overnight, quenchedwith methanol (10 mL) before diluting with ethyl acetate (1 L) and water(500 mL). The organic layer was washed with water (3×500 mL) andconcentrated to a solid. The solid was recrystallized from hottert-butyl methyl ether (300 mL) to give p-thiophenyl2,3-di-O-benzyl-4,6-O-benzylidene-β-D-glucopyranoside (86 g).

p-Thiophenyl 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-glucopyranoside (8.4g, 15 mmol) was dissolved in CH₂Cl₂ (300 mL), a solution oftrifluoroacetic acid (8.4 mL) in water (5.6 mL) was added, and theresulting mixture was stirred at room temperature for 4 hours. Thereaction was quenched with saturated NaHCO₃ solution (150 mL), and theorganic layer was dried over Na₂SO₄, filtered and concentrated. Thecrude residue was purified by dissolving in a minimum volume of CH₂Cl₂and passing it through a silica plug, eluting with ethyl acetate:heptane(1:9) then ethyl acetate:heptane (3:2) to give p-thiophenyl2,3-di-O-benzyl-β-D-glucopyranoside (5.3 g, 75%).

p-Thiophenyl 2,3-di-O-benzyl-β-D-glucopyranoside (6.0 g, 13 mmol) wasdissolved in dry NMP:THF (1:4, 100 mL) and cooled to 0° C. withstirring. Solid NaH (1.55 g of 60% suspension, 39 mmol) was added slowlyand the mixture was stirred for 15 minutes. Benzyl bromide (3.8 mL, 32mmol) was added slowly to keep the internal temperature below 20° C. Thereaction mixture was stirred at room temperature overnight, quenchedwith methanol (10 mL) before diluting with ethyl acetate (150 mL). Theorganic layer was washed with 1 N HCl (100 mL), brine (100 mL) andsaturated NaHCO₃ solution (100 mL), dried over MgSO₄, filtered andconcentrated. The solid was purified on a silica gel column (120 g)using an ISCO automated chromatography system, eluting with a 0→40%gradient of ethyl acetate in heptane, to give the desired p-thiophenyl2,3,4,6-tetra-O-benzyl-β-D-glucopyranoside 6 (4.66 g, 55%).

p-Thiophenyl 2,3,4,6-tetra-O-benzyl-β-D-glucopyranoside 6 (4.66 g, 7.2mmol) was co-evaporated with toluene (2×10 mL) then dissolved in dryCH₂Cl₂ (50 mL) under nitrogen and cooled to 0° C. Bromine (465 μL, 18mmol) was added and the reaction mixture stirred for 15 minutes. Thereaction was quenched with saturated Na₂S₂O₃ solution (50 mL), and theaqueous layer was extracted with CH₂Cl₂ (3×50 mL). The combined organicextracts were dried over Na₂SO₄, filtered and concentrated to give thecrude glucosyl bromide, which was used without further purification.

The glucosyl bromide and the tetrasaccharide diol acceptor 4 (2.0 g, 1.2mmol) were combined and co-evaporated with toluene (2×10 mL), taken upin anhydrous diethyl ether (50 mL) under nitrogen and cooled to −50° C.Silver triflate (1.85 g, 7.2 mmol) was added and the reaction mixturewas stirred for I hour. The reaction was quenched by the addition ofsaturated sodium ascorbate solution (25 mL) and stirring for 1 hour. Themixture was filtered through celite and concentrated to dryness. Thecrude residue was purified by size exclusion chromatography using BioRadSX-1 Bio-Beads (4 cm×40 cm column) and toluene as the eluent. Thefractions containing hexasaccharide were further purified on a silicagel column (40 g) using an ISCO automated chromatography system, elutingwith a 0→80% gradient of ethyl acetate in heptane, to give the desiredhexasaccharide 7 (2.6 g, 79%).

Hexasaccharide 7 (1.05 g, 0.383 mmol) was co-evaporated with toluene (10mL), dissolved in dioxane (12 mL) under N₂, and thioacetic acid (1.0 mL,14.2 mmol) was added. The solution was purged with nitrogen gas for 15minutes before AlBN (100 mg, 0.61 mmol) was added and the reactionmixture heated to 80° C. under an inert atmosphere for 3 hours thenovernight at room temperature. The solvent was removed and the crudeproduct was co-evaporated with toluene. The crude residue was purifiedby size exclusion chromatography using BioRad SX-1 Bio-Beads (2.5 cm×25cm column) and toluene as the eluent. The fractions containing purifiedhexasaccharide were further purified on a silica gel column (40 g) usingan ISCO automated chromatography system, eluting with a 0→75% gradientof ethyl acetate in heptane, to give the desired hexasaccharide (350 mg,32%). Mixed fractions were reprocessed through Bio-Beads and silica gelchromatography to yield more of the desired hexasaccharide thioacetateproduct.

Hexasaccharide thioacetate (330 mg, 0.117 mmol) was coevaporated withtoluene (2×10 mL) and dissolved in dry THF (5 mL). In a jacketed flaskequipped with a dry ice condenser and cooled to −78° C., NH₃ gas wascondensed into the flask under an Ar atmosphere until the liquid volumereached approximately 20 mL. The hexasaccharide thioacetate solution wastransferred by cannula into the liquid NH₃. Sodium metal (250 mg, 10.9mmol) was added under positive Ar gas flow and once a permanent bluecolor was established the reaction was stirred for another 20 minutes.The reaction was quenched at −78° C. by the addition of saturated NH₄Clsolution (10 mL) and warmed to room temperature. N₂ was bubbled throughthe reaction mixture to remove excess NH₃, and then the solution wasconcentrated to 2 mL. The crude material was purified by size exclusionchromatography on a bed of BioGel P4 (2.5 cm×50 cm column) by elutionwith water. The fractions containing product were concentrated to yieldthe desired Moraxella Serotype B7 hexasaccharide core 8 (104 mg, 83%).

Synthesis of Serotype B7 Hexasaccharide Core Conjugates 9, 41 (FIG. 8B)

To synthesize the KLH conjugate 9, a solution oftris(2-carboxyethyl)phosphine (TCEP) in water (8.6 μL, 0.05 M, 0.43μmol) was added to a solution of hexasaccharide thiol 8 in water (15.6mg/mL, 58 μL, 0.86 μmol), and the resulting solution was stirred for 1hour. A solution of maleimide-activated keyhole limpet hemocyanin(Imject® KLH, Pierce, Rockford, Ill.) (0.5 mL, 5 mg, ˜0.43 μmolmaleimide) was added and the resulting solution stirred overnight atroom temperature. The reaction mixture was purified by de-salting onD-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). The column waspre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.77159), the crude material loaded onto the column and eluted withpurification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired serotype B7hexamer-KLH conjugate 9.

To synthesize the BSA conjugate 41, a solution oftris(2-carboxyethyl)phosphine (TCEP) in water (30 μL, 0.05 M, 1.5 μmol)was added to a solution of hexasaccharide thiol 8 in water (15.6 mg/mL,205 μL, 3.0 μmol), and the resulting solution was stirred for 1 hour. Asolution of maleimide-activated bovine serum albumin (Imject® BSA,Pierce, 5 mg, ˜1.5 μmol maleimide) in Imject® Conjugation Buffer(Pierce, 250 μL diluted with water, 250 μL) was added and the resultingsolution stirred overnight at room temperature. The reaction mixture waspurified by de-salting on D-Salt P-6000 10 mL column (Pierce, Rockford,Ill.). The column was pre-equilibrated with 30 mL of purification buffer(Pierce, Prod. No. 77159), the crude material loaded onto the column andeluted with purification buffer. 1-mL fractions were collected andanalyzed for protein content by absorbance at 280 nm (A₂₈₀). Fractionscontaining protein were combined and lyophilized to give the desiredserotype B7 hexamer-BSA conjugate 41.

Example 6 Synthesis of Serotype B9 Octamer and Conjugates ThereofSynthesis of Serotype B9 Octamer 10 (FIG. 9)

Thiomethyl4-O-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-β-D-glucopyranoside51 (2.76 g, 2.95 mmol) (J. Org. Chem. 61 (1996) 7711) was co-evaporatedwith toluene (2×10 mL) then dissolved in dry CH₂Cl₂ (30 mL) undernitrogen and cooled to 0° C. Bromine (191 μL, 7.4 mmol) was added andthe reaction mixture stirred for 10 minutes. The reaction was quenchedwith saturated Na₂S₂O₃ solution (50 mL), and the aqueous layer wasextracted with CH₂Cl₂ (3×50 mL). The combined organic extracts weredried over Na₂SO₄, filtered and concentrated to give the crude glucosylbromide, which was used without further purification.

The glycosyl bromide and the tetrasaccharide diol acceptor 4 (0.50 g,0.30 mmol) were combined and co-evaporated with toluene (2×10 mL), takenup in anhydrous diethyl ether (10 mL) under nitrogen and cooled to −40°C. Silver triflate (0.760 g, 2.95 mmol) was added and the reactionmixture was stirred for I hour. The reaction was quenched by pouringinto saturated sodium ascorbate solution (25 mL) and stirring for 30minutes. The mixture was filtered through celite and the filtrate washedwith ethyl acetate (3×50 mL). The organic layer was washed with brine,dried over Na₂SO₄, filtered and concentrated. The crude residue waspurified by size exclusion chromatography using BioRad SX-1 Bio-Beads(2.5 cm×50 cm column) and toluene as the eluent. The fractionscontaining octasaccharide were further purified twice on a silica gelcolumn (80 g) using an ISCO automated chromatography system, elutingwith a 0→20% gradient of ethyl acetate in heptane, to give the desiredoctasaccharide 10 (0.250 g, 24%).

Octasaccharide 10 (0.25 g, 0.069 mmol) was dissolved in dioxane (6 mL),thioacetic acid (0.5 mL, 7.1 mmol) was added, and the solution purgedwith argon gas for 10 minutes. The solution was purged with nitrogen gasfor 15 minutes before AlBN (50 mg, 0.30 mmol) was added and the reactionmixture heated to 75° C. under an inert atmosphere overnight. A secondaliquot of AlBN (25 mg, 0.15 mmol) was added and the reaction mixturewas heated until the reaction was complete. The solvent was removed andthe crude product was co-evaporated with toluene (2×10 mL). The cruderesidue was purified by size exclusion chromatography using BioRad SX-1Bio-Beads (2.5 cm×25 cm column) and toluene as the eluent. Appropriatefractions were combined to give the desired octasaccharide thioacetateproduct (225 mg, 88%).

Octasaccharide thioacetate (220 mg, 0.060 mmol) was co-evaporated withtoluene (2×10 mL) and dissolved in dry THF (5 mL). In a flame-driedjacketed flask equipped with a dry ice condenser and cooled to −78° C.,NH₃ gas was condensed into the flask under an Ar atmosphere until theliquid volume reached approximately 10 mL. The octasaccharidethioacetate solution was transferred by cannula into the liquid NH₃.Sodium metal (250 mg, 10.9 mmol) was added under positive Ar gas flowand once a permanent blue color was established the reaction was stirredfor another 20 minutes. The reaction was quenched at −78° C. by theaddition of saturated NH₄Cl solution (10 mL) and warmed to roomtemperature. N₂ was bubbled through the reaction mixture to removeexcess NH₃, and then the solution was concentrated to dryness. The crudematerial was purified by size exclusion chromatography on a bed ofBioGel P4 (2.5 cm×60 cm column) by elution with water. The fractionscontaining product were concentrated to yield the desired Moraxellaserotype B9 octasaccharide 11 (20 mg, 24%).

Synthesis of Serotype B9 Octamer Conjugates 12, 40 (FIG. 9)

To synthesize the KLH conjugate 12, a solution oftris(2-carboxyethyl)phosphine (TCEP) in water (8.6 μL, 0.05 M, 0.43μmol) was added to a solution of octasaccharide thiol 11 in water (15.4mg/mL, 79 μL, 0.86 μmol), and the resulting solution was stirred for 1hour. A solution of maleimide-activated keyhole limpet hemocyanin(Imject® KLH, Pierce, Rockford, Ill.) (0.5 mL, 5 mg, ˜0.43 μmolmaleimide) was added and the resulting solution stirred overnight atroom temperature. The reaction mixture was purified by de-salting onD-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). The column waspre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.77159), the crude material loaded onto the column and eluted withpurification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired serotype B9octamer-KLH conjugate 12.

To synthesize the BSA conjugate 40, a solution oftris(2-carboxyethyl)phosphine (TCEP) in water (30 μL, 0.05 M, 1.5 μmol)was added to a solution of octasaccharide thiol 11 in water (15.4 mg/mL,276 μL, 3.0 μmol), and the resulting solution was stirred for 1 hour. Asolution of maleimide-activated bovine serum albumin (Imject® BSA,Pierce, 5 mg, ˜1.5 μmol maleimide) in Imject® Conjugation Buffer(Pierce, 250 μL diluted with water, 250 μL) was added and the resultingsolution stirred overnight at room temperature. The reaction mixture waspurified by de-salting on D-Salt P-6000 10 mL column (Pierce, Rockford,Ill.). The column was pre-equilibrated with 30 mL of purification buffer(Pierce, Prod. No. 77159), the crude material loaded onto the column andeluted with purification buffer. 1-mL fractions were collected andanalyzed for protein content by absorbance at 280 nm (A₂₈₀). Fractionscontaining protein were combined and lyophilized to give the desiredserotype B9 octamer-BSA conjugate 40.

Example 7 Synthesis of Serotype B11 Decamer and Conjugates ThereofSynthesis of Serotype B11 Decamer 20 (FIGS. 10A, 10B)

With reference to FIG. 10A, allyl3-O-(3,4,6-tri-O-benzyl-β-D-glucopyranosyl)-4,6-O-benzylidene-α-D-glucopyranoside57 (33.5 g, 45.2 mmol) was co-evaporated with toluene (2×25 mL) beforebeing taken up in a 4:1 solution of NMP:THF (400 mL) and cooled to 0° C.Solid NaH (4.52 g of 60% suspension, 113 mmol) was added and the mixturewas stirred for 10 minutes before the slow addition of benzyl bromide(12.9 mL, 146 mmol) over 20 minutes. The reaction mixture was stirred atroom temperature for 18 hours, quenched with methanol, stirred for 1hour and the solvent removed. The residue was suspended in ethyl acetate(1 L), washed with 1 N HCl, brine, and saturated NaHCO₃ (250 mL each).The ethyl acetate solution was dried over MgSO₄, filtered andconcentrated in vacuo. The crude material was used without furtherpurification.

The crude benzylation product was taken up in CH₂Cl₂ (200 mL) and asolution of trifluoroacetic acid in water (3:2, 10 mL) was added. Thereaction mixture was stirred for 16 hours then quenched with solidNaHCO₃ and stirred until bubbling ceased. The layers were separated andthe aqueous phase was extracted with CH₂Cl₂ (200 mL). The combinedorganic extracts were dried over MgSO₄, filtered and concentrated invacuo. The crude residue was passed through a silica plug, eluting with9:1 heptane: ethyl acetate to remove benzaldehyde-related material, thenwith 1:1 ethyl acetate: CH₂Cl₂. The residue was crystallized fromethanol to give allyl3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2-O-benzyl-α-D-glucopyranoside14 (22.2 g, 67%).

Allyl3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2-O-benzyl-α-D-glucopyranoside14 (10.1 g, 12.1 mmol), 4-methoxyphenol (3.76 g, 30.3 mmol), andtriphenylphosphine (4.77 g, 18.2 mmol) were combined in a flask, cooledto 0° C. before adding anhydrous CH₂Cl₂ (100 mL). This solution wasstirred for 40 minutes and then a solution of diethylazodicarboxylate(DEAD) in toluene (40% by weight, 8.84 mL, 19.4 mmol) was added. Thereaction mixture was stirred at 0° C. for 45 minutes, and then allowedto warm to room temperature overnight. The reaction mixture was dilutedwith CH₂Cl₂, washed with 1N NaOH (200 mL, 100 mL), 1 N HCl (200 mL),saturated NaHCO₃ (200 mL), and brine, dried over Na₂SO₄, filtered andconcentrated. The residue was purified on a silica gel column elutedusing an ethyl acetate: heptane step gradient (1:10→1:5→1:3) to giveallyl3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2-O-benzyl-6-O-(4-methoxyphenyl)-α-D-glucopyranoside14 (7.81 g, 69%).

Allyl3-O-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2-O-benzyl-6-O-(4-methoxyphenyl)-α-D-glucopyranoside14 (2.62 g, 2.79 mmol) and2-pivaloyl,3,4,6-tri-O-benzyl-β-D-glucopyranosyl trichloroacetimidate 2(2.85 g, 4.20 mmol) were combined, co-evaporated with toluene (3×10 mL)and dissolved in dry CH₂Cl₂ (25 mL). Freshly activated AW-300 molecularsieves (3 g) were added and the reaction mixture was stirred for 10minutes and cooled to 0° C. A solution of trimethylsilyltrifluoromethanesulfonate (TMSOTf, 0.05 mL, 0.28 mmol) in CH₂Cl₂ (0.45mL) was added and the reaction stirred for 30 minutes, at which pointanother aliquot of 2 (0.95 g, 1.4 mmol) was added. After another 30minutes the reaction mixture was quenched with Et₃N (1 mL), filtered andthe solvent removed to give the crude coupling product, which waspurified by silica gel chromatography (500 mL column), eluting with 3:1heptane: ethyl acetate to give the desired trisaccharide (3.5 g, 86%).

The trisaccharide coupling product (1.75 g, 1.20 mmol) was dissolved inmethanol: tetrahydrofuran (2:1, 15 mL) and sodium methoxide solution(1.0 mL, 25% by weight, 4.4 mmol) was added. The solution was stirred atroom temperature for 42 hours, quenched with 1N HCl, diluted with ethylacetate (200 mL), washed with brine (100 mL), and saturated NaHCO₃ (100mL), dried over Na₂SO₄, filtered and concentrated. The crude materialwas purified on a silica gel column (40 g) using an ISCO automatedchromatography system, eluting with a 0→70% gradient of ethyl acetate inheptane, to give the desired trisaccharide 15 (0.870 g, 50%).

Thiomethyl4-O-(2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-β-D-glucopyranoside16 (2.50 g, 1.75 mmol) (J. Org. Chem. 61 (1996) 7711) was co-evaporatedwith toluene (2×10 mL) then dissolved in dry CH₂Cl₂ (20 mL) undernitrogen and cooled to 0° C. Bromine (115 μL, 4.5 mmol) was added andthe reaction mixture stirred for 20 minutes. The reaction was quenchedwith saturated Na₂S₂O₃ solution (50 mL), and the aqueous layer wasextracted with CH₂Cl₂ (3×50 mL). The combined organic extracts weredried over Na₂SO₄, filtered and concentrated to give the crude glycosylbromide, which was used without further purification.

The glycosyl bromide and the trisaccharide acceptor 15 (825 mg, 0.60mmol) were combined and co-evaporated with toluene (2×10 mL), taken upin anhydrous diethyl ether (10 mL) under nitrogen and cooled to −40° C.Silver triflate (450 mg, 1.75 mmol) was added and the reaction mixturewas stirred for 1 hour. The reaction was quenched by pouring intosaturated sodium ascorbate solution (25 mL) and stirring for 30 minutes.The mixture was filtered through celite and the filtrate washed withethyl acetate (3×50 mL). The organic layer was washed with brine, driedover Na₂SO₄, filtered and concentrated. The crude residue was passedthrough a silica plug, eluting with heptane: ethyl acetate, the solventwas removed and the crude hexasaccharide was used without furtherpurification.

The crude hexasaccharide (˜0.6 mmol) was taken up in acetonitrile andwater (4:1, 20 mL) and cooled to 0° C. Ammonium cerium(IV) nitrate (CAN,1.15 g, 2.1 mmol) was added and the reaction mixture was stirred for 2hours. Ethyl acetate (50 mL) was added and the mixture was washed withbrine and saturated NaHCO₃, (2×50 mL), dried over Na₂SO₄, filtered andconcentrated. The crude residue was purified by silica gelchromatography eluting with 15% ethyl acetate in toluene to give thedesired hexasaccharide 17 (280 mg, 18%).

Hexasaccharide acceptor 17 (333 mg, 0.126 mmol) and2-pivaloyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl trichloroacetimidate 2(171 mg, 0.251 mmol) were combined and dissolved in dry CH₂Cl₂ (5 mL).Freshly activated AW-300 molecular sieves (300 mg) were added and thereaction mixture was stirred for 20 minutes and cooled to 0° C.Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 5 μL, 0.0125 mmol) wasadded and the reaction stirred for 30 minutes, at which point anotheraliquot of 2 (0.95 g, 1.4 mmol) was added. After another 30 minutes thereaction mixture was quenched with Et₃N (1 mL), diluted with CH₂Cl₂ (5mL), filtered and concentrated. The crude coupling product was purifiedby silica gel chromatography, eluting with a heptane: ethyl acetate stepgradient (5:1→4:1→3:1) to give the desired intermediate heptasaccharide(298 mg, 75%).

The heptasaccharide (620 mg, 0.200 mmol) was dissolved inmethanol:tetrahydrofuran (1:1, 20 mL) and sodium methoxide solution (2.0mL, 25% by weight, 8.8 mmol) was added. The solution was stirred at 45°C. for 43 hours, cooled to room temperature and concentrated to ˜5 mL.The residue was dissolved in ethyl acetate (50 mL), washed with 1N HCl(50 mL), saturated NaHCO₃ (50 mL) and brine (50 mL), dried over Na₂SO₄,filtered and concentrated. The crude material was purified by silica gelchromatography, eluting with a heptane: ethyl acetate step gradient togive the desired heptasaccharide 18 (570 mg, 94%).

Turning to FIG. 10B, thiomethyl4-O-(2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-β-D-glucopyranoside16 (745 mg, 0.519 mmol) (J. Org. Chem. 61 (1996) 7711) was dissolved indry CH₂Cl₂ (8.5 mL) under nitrogen and cooled to 0° C. Bromine (66 μL,1.28 mmol) was added and the reaction mixture stirred for 20 minutes.The reaction was quenched with saturated Na₂S₂O₃ solution (25 mL), andstirred until the color faded, then was diluted with CH₂Cl₂ (50 mL). Theorganic layer was washed with saturated NaHCO₃ (50 mL) and brine (50mL), dried over Na₂SO₄, filtered and concentrated to give the crudeglycosyl bromide, which was used without further purification.

The glycosyl bromide and the heptasaccharide acceptor 18 (525 mg, 0.170mmol) were combined and dried under vacuum, 4 Å molecular sieves (700mg) were added and anhydrous diethyl ether (10 mL) was added undernitrogen. The mixture was cooled to −40° C. before silver triflate (140mg, 0.545 mmol) was added and the reaction mixture was stirred for 1hour. The reaction was warmed to −20° C. over 30 minutes then quenchedby pouring into saturated sodium ascorbate solution (25 mL) and stirringfor 30 minutes. The mixture was filtered through celite and the filtratewashed with saturated NaHCO₃ (50 mL) and brine (50 mL), dried overNa₂SO₄, filtered and concentrated. The crude residue was purified bysilica gel chromatography, eluting with a toluene: ethyl acetate stepgradient (20:1→18:1→15:1) to give enriched decasaccharide. The partiallypurified decasaccharide was purified to homogeneity by size exclusionchromatography using BioRad SX-1 BioBeads eluting with toluene (two2.5×50 cm columns) followed by another silica gel column eluting withheptane: ethyl acetate 2:1 to give the desired decasaccharide 19 (567mg, 74%).

Decasaccharide 19 (400 mg, 0.089 mmol) was dissolved in dioxane (6 mL),thioacetic acid (0.5 mL, 7.1 mmol) was added, and the solution purgedwith argon gas for 10 minutes. The solution was purged with nitrogen gasfor 15 minutes before AlBN (50 mg, 0.30 mmol) was added and the reactionmixture heated to 75° C. under an inert atmosphere for 18 hours. Thesolvent was removed and the crude product was co-evaporated with toluene(2×10 mL). The crude residue was purified by size exclusionchromatography using BioRad SX-1 Bio-Beads (2.5 cm×25 cm column) andtoluene as the eluent. Appropriate fractions were combined to give thedesired octasaccharide thioacetate product (370 mg, 91%).

Decasaccharide thioacetate (370 mg, 0.081 mmol) was co-evaporated withtoluene (2×10 mL) and dissolved in dry THF (5 mL). In a flame-driedjacketed flask equipped with a dry ice condenser and cooled to −78° C.,NH₃ gas was condensed into the flask under an Ar atmosphere until theliquid volume reached approximately 10 mL. The decasaccharidethioacetate solution was transferred by cannula into the liquid NH₃.Sodium metal (200 mg, 8.7 mmol) was added under positive Ar gas flow andonce a permanent blue color was established the reaction was stirred foranother 20 minutes. The reaction was quenched at −78° C. by the additionof saturated NH₄Cl solution (10 mL) and warmed to room temperature. N₂was bubbled through the reaction mixture to remove excess NH₃, and thenthe solution was concentrated to dryness. The crude material waspurified by size exclusion chromatography on a bed of BioGel P4 (2.5cm×60 cm column) by elution with water. The fractions containing productwere concentrated to yield the desired Moraxella serotype B11decasaccharide 20 (92 mg, 30%).

Synthesis of Serotype B11 Decamer Conjugates 21, 22 (FIG. 10B)

To synthesize the KLH conjugate 21, a solution oftris(2-carboxyethyl)phosphine (TCEP) in water (8.6 μL, 0.05 M, 0.43μmol) was added to a solution of decasaccharide thiol 20 in water (12.8mg/mL, 110 μL, 0.86 μmol), and the resulting solution was stirred for 1hour. A solution of maleimide-activated keyhole limpet hemocyanin(Imject® KLH, Pierce, Rockford, Ill.) (0.5 mL, 5 mg, 0.43 μmolmaleimide) was added and the resulting solution stirred overnight atroom temperature. The reaction mixture was purified by de-salting onD-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). The column waspre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.77159), the crude material loaded onto the column and eluted withpurification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired serotype B11decamer-KLH conjugate 21.

To synthesize the BSA conjugate 22, a solution oftris(2-carboxyethyl)phosphine (TCEP) in water (30 μL, 0.05 M, 1.5 μmol)was added to a solution of decasaccharide thiol 20 in water (12.8 mg/mL,390 μL, 3.0 μmol), and the resulting solution was stirred for 1 hour. Asolution of maleimide-activated bovine serum albumin (Imject® BSA,Pierce, 5 mg, ˜1.5 μmol maleimide) in Imject® Conjugation Buffer(Pierce, 250 μL diluted with water, 250 μL) was added and the resultingsolution stirred overnight at room temperature. The reaction mixture waspurified by de-salting on D-Salt P-6000 10 mL column (Pierce, Rockford,Ill.). The column was pre-equilibrated with 30 mL of purification buffer(Pierce, Prod. No. 77159), the crude material loaded onto the column andeluted with purification buffer. 1-mL fractions were collected andanalyzed for protein content by absorbance at 280 nm (A₂₈₀). Fractionscontaining protein were combined and lyophilized to give the desiredserotype B11 decamer-BSA conjugate 22.

Example 8 Synthesis of Serotype C11 Decamer and Conjugates ThereofSynthesis of Serotype C11 Decamer 37 (FIGS. 11A, 11B)

With reference to FIG. 11A,1,6-anhydro-2-deoxy-2-azido-3-O-benzyl-β-D-glucopyranose 31 (765 mg,2.75 mmol) and2-benzoyl-4-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-3,6-di-O-benzyl-D-galactopyranosyltrichloroacetimidate 30 (2.3 mmol) were combined, co-evaporated withtoluene (2×10 mL) and dissolved in dry CH₂Cl₂ (30 mL). Freshly activatedAW-300 molecular sieves (2 g) were added and the reaction mixture wasstirred for 10 minutes and cooled to 0° C. A solution of trimethylsilyltrifluoromethanesulfonate (TMSOTf, 0.042 mL, 0.23 mmol) in CH₂Cl₂ (0.38mL) was added and the reaction stirred for 30 minutes. The reactionmixture was quenched with Et₃N (0.5 mL), filtered and the solventremoved to give the crude coupling product, which was purified on asilica gel column (40 g) using an ISCO automated chromatography system,eluting with a 0→40% gradient of ethyl acetate in heptane, to give thedesired trisaccharide 32 (1.9 g, 66%).

Acetic anhydride (15 mL) was added to trisaccharide 32 (1.8 g, 1.43mmol), and Sc(OTf)₃.H₂O (30 mg, 0.058 mmol) was added. After 5 hours asecond aliquot of Sc(OTf)₃.H₂O (30 mg, 0.058 mmol) was added and thereaction mixture was stirred overnight. The reaction mixture was cooledto 0° C. and methanol (10 mL) was added over 30 minutes, keeping thetemperature below 10° C. The mixture was partitioned between ethylacetate and water (100 mL each). The organic layer was washed with water(3×50 mL), saturated NaHCO₃ (50 mL) and brine (50 mL), dried overNa₂SO₄, filtered and concentrated. The crude material was purified on asilica gel column (80 g) using an ISCO automated chromatography system,eluting with a 0→70% gradient of ethyl acetate in heptane, to give amixture of trisaccharides (1.8 g, >90%). The mixture was predominantlythe desired 1,6-di-O-acetate containing product, but also included smallamounts of similar compounds in which the other primary benzyl ethershad been exchanged for acetyl groups. The mixture was used withoutfurther purification.

The trisaccharide mixture (1.8 g, 1.3 mmol) was taken up in dry THF (20mL) and purged with nitrogen for 10 minutes while cooling to 0° C.Ammonia gas was bubbled through the solution for 15 minutes and thereaction mixture was stirred for 2 hours, during which the temperaturewas allowed to return to room temperature. The solution was purged withnitrogen for 30 minutes and then concentrated in vacuo. The residue waspurified on a silica plug, eluting with heptane: ethyl acetate (1:1) togive the lactol as a yellow oil (1.6 g, 92%).

The trisaccharide lactol mixture (1.6 g, 1.31 mmol) was dissolved inCH₂Cl₂ (25 mL) and trichloroacetonitrile (5 mL) was added. Solid K₂CO₃(2 g) was added and the heterogeneous mixture was stirred at roomtemperature for 2 hours before filtering through celite. The filtratewas concentrated, redissolved in heptane:ethyl acetate (1:1) andfiltered through a 30-mL silica plug that had been pre-washed withheptane:ethyl acetate:Et3N (1:1:0.01→1:1:0). The solvent was removed tothe desired trisaccharide trichloroacetimidate 33 (1.5 g, 84%).

The trisaccharide trichloroacetimidate donor 33 (1.5 g, 1.1 mmol) andthe trisaccharide acceptor 15 were combined, co-evaporated with toluene(3×10 mL) and dissolved in dry ether (20 mL). Freshly activated 4 ÅAW-300 molecular sieves (2 g) were added and the reaction mixture wasstirred for 1 hour and cooled to 0° C. A solution of trimethylsilyltrifluoromethanesulfonate (TMSOTf) in ether (0.1 M, 1.1 mL) was addedover 5 minutes and the reaction stirred for 1 hour at 0° C., at whichpoint another 0.1 equivalents of TMSOTf was added. After another 1 hourthe reaction mixture was quenched with Et₃N (0.2 mL), filtered throughcelite and the solvent removed to give the crude coupling product, whichwas purified on a silica gel column (40 g) using an ISCO automatedchromatography system, eluting with a 0→70% gradient of ethyl acetate inheptane, to give a mixture of hexasaccharides, which included α- andβ-anomers (2.2 g, 78%).

The hexasaccharide mixture (2.2 g, 0.85 mmol) was dissolved in methanol:tetrahydrofuran (1:1, 20 mL) and sodium methoxide solution (1.0 mL, 25%by weight, 4.4 mmol) was added. The solution was stirred at roomtemperature for 18 hours, at which point another aliquot of sodiummethoxide solution (1.0 mL, 25% by weight, 4.4 mmol) was added. Thesolution was stirred at room temperature for an additional 2 hours,quenched with A-15(H⁺) resin and filtered through celite. Concentrationof the filtrate gave the crude hexasaccharide polyol, which was usedwithout further purification.

The crude hexasaccharide polyol (˜0.85 mmol) was taken up in a 4:1solution of NMP:THF (24 mL) and cooled to 0° C. Benzyl bromide (0.71 mL,5.95 mmol) was added followed by solid NaH (0.27 g of 60% suspension,6.8 mmol). The mixture was stirred for 4 hours, over which the mixturewarmed to room temperature, and additional aliquots of NaH (0.27 g) andBnBr (0.71 mL) were added. After stirring an additional 18 hours thereaction was quenched with methanol (5 mL) and the solvent removed. Theresidue was suspended in ethyl acetate (100 mL), washed with water andbrine, dried over Na₂SO₄, filtered and concentrated in vacuo. The crudematerial was purified on a silica gel column (120 g) using an ISCOautomated chromatography system, eluting with a 0→50% gradient of ethylacetate in heptane, to give a mixture of hexasaccharides, which includedα- and β-anomers. A second silica gel purification (80 g column) usingan ISCO automated chromatography system, eluting with a 0→40% gradientof ethyl acetate in heptane, gave the desired hexasaccharide as pureα-anomer (1.2 g).

The crude hexasaccharide (1.14 g, ˜0.42 mmol) was taken up inacetonitrile and water (4:1, 15 mL) and cooled to 0° C. Ammoniumcerium(IV) nitrate (CAN, 700 mg, 1.27 mmol) was added and the reactionmixture was stirred for 2 hours. The reaction was quenched with Et₃N (3mL) and filtered through celite, washing with CH₂Cl₂ (75 mL). Thesolution was dried over Na₂SO₄, filtered and concentrated. The cruderesidue was purified by silica gel chromatography eluting with aheptane: ethyl acetate step gradient (4:1→3:1→2:1→1:1) to give thedesired hexasaccharide 34 (752 mg, 69%) and recovered starting material(210 mg, 18%).

Hexasaccharide acceptor 34 (750 mg, 0.290 mmol) and di-n-butylphosphoryl2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranoside 55 (238 mg, 0.348mmol) were combined, co-evaporated with toluene (3×5 mL), dissolved indry CH₂Cl₂ (6 mL) and cooled under nitrogen to −30° C. The reactionmixture was stirred for 10 minutes before trimethylsilyltrifluoromethanesulfonate (TMSOTf, 63 μL, 0.348 mmol) was added. Thereaction mixture was stirred for 20 minutes then quenched with Et₃N (4mL), and the solvent removed to give the crude coupling product, whichwas purified by silica gel chromatography (250 mL column), eluting usinga heptane:ethyl acetate step gradient (6:1→4:1→3:1→2:1) to give thedesired heptasaccharide coupling product (750 mg, 84%).

Heptasaccharide 35 (750 mg, 0.245 mmol) was dissolved inmethanol:tetrahydrofuran (2:1, 15 mL) and sodium methoxide solution (2.0mL, 25% by weight, 8.8 mmol) was added. The solution was stirred at roomtemperature for 4 hours and concentrated in vacuo. The residue waspartitioned between ethyl acetate (150 mL) and saturated NH₄Cl (100 mL),washed with brine (2×100 mL), dried over Na₂SO₄, filtered andconcentrated. The crude material was purified by silica gelchromatography (250 mL column), eluting using a heptane: ethyl acetatestep gradient (4:1→2:1) to give the desired heptasaccharide 35 (719 mg,97%).

Thiomethyl4-O-(2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-β-D-glucopyranoside16 (779 mg, 0.541 mmol) (J. Org. Chem. 61 (1996) 7711) was co-evaporatedwith toluene (3×8 mL) then dissolved in dry CH₂Cl₂ (5 mL) under nitrogenand cooled to 0° C. Bromine (29 μL, 0.568 mmol) was added and thereaction mixture stirred for 10 minutes. Toluene (10 mL) was added, themixture concentrated to dryness and the toluene azeotrope step wasrepeated twice more to give the crude glycosyl bromide, which was usedwithout further purification.

The heptaccharide acceptor 35 (543 mg, 0.180 mmol) was co-evaporatedwith toluene (3×8 mL). Freshly activated 4 Å molecular sieves (1 g) wereadded under nitrogen. The glycosyl bromide was taken up in anhydrousdiethyl ether (10 mL) under nitrogen, transferred to theacceptor/molecular sieves mixture and cooled to −40° C. Silver triflate(139 mg, 0.541 mmol) was added and the reaction mixture was stirred for1 hour. The reaction was quenched at −20° C. with Et₃N (0.4 mL), dilutedwith CH₂Cl₂ (50 mL) and filtered through celite. The filtrate wasstirred with saturated sodium ascorbate solution (50 mL) for 20 minutes,filtered through celite and the organic layer of the filtrate was washedwith saturated NaHCO3 (100 mL) and brine (100 mL), dried over Na₂SO₄,filtered and concentrated. The crude residue was purified by silica gelchromatography (250 mL column), eluting with a toluene: ethyl acetatesolvent system, and then by size exclusion chromatography on BioRad SX-1BioBeads, eluting with toluene to give the desired decasaccharide 36(430 mg, 54%).

Turning now to FIG. 11B, the decasaccharide 36 (400 mg, 0.091 mmol) wasdissolved in dioxane (3 mL), AlBN (40 mg, 0.24 mmol) was added and thesolution purged with nitrogen gas. Thioacetic acid (0.3 mL, 4.3 mmol)was added, and the solution purged with nitrogen gas for 15 minutes andthe reaction mixture heated to 80° C. under an inert atmosphere for 18hours. The solution was cooled to room temperature, quenched withcyclohexene (0.2 mL) and concentrated in vacuo. The crude residue waspurified by size exclusion chromatography using BioRad SX-1 Bio-Beads(2.5 cm×60 cm column) and toluene as the eluent. Appropriate fractionswere combined to give the desired decasaccharide thioacetate product(260 mg, 64%) mixed with recovered starting material (20%). The mixturewas re-subjected to the reaction conditions and purification scheme toconvert the remaining starting material to the desired decasaccharidethioacetate (300 mg, 74% total yield).

The decasaccharide thioacetate (162 mg, 0.036 mmol) was dissolved in DMF(3 mL) and ethanol (1.5 mL), and the solution was purged with nitrogenfor 5 minutes. Benzyl chloride (60 μL, 0.52 mmol) and 1.0 M NaOH (0.2mL) were added, and the reaction mixture was stirred for 1 hour. Themixture was diluted with ethyl acetate (50 mL), washed with water (2×50mL) and brine (2×50 mL), dried over Na₂SO₄, filtered and concentrated toa yellow oil (150 mg), which was used without further purification.

Decasaccharide benzyl thioether (150 mg, 0.033 mmol) was dissolved indry THF (2 mL). In a flame-dried jacketed flask equipped with a dry icecondenser and cooled to −78° C., NH₃ gas was condensed into the flaskunder an Ar atmosphere until the liquid volume reached approximately 15mL. The decasaccharide benzyl thioether solution was transferred intothe liquid NH₃. Sodium metal (20 mg, 0.87 mmol) was added under positiveAr gas flow and stirred for 10 minutes. The reaction was quenched at−78° C. by the addition of saturated NH₄Cl solution (0.1 mL) and warmedto room temperature. N₂ was bubbled through the reaction mixture toremove excess NH₃, and then the solution was concentrated to dryness.The crude material was purified by size exclusion chromatography on abed of Sephadex G-10 (2.5 cm×8 cm column) by elution with water. Thefractions containing product were concentrated to yield the crudedecasaccharide with co-eluting salts (120 mg).

The crude decasaccharide/salt mixture (120 mg) was dissolved in water(0.8 mL) and methanol (0.4 mL), solid NaHCO₃ (40 mg), and aceticanhydride (40 μL) were added. The reaction mixture was stirred for 2hours then loaded directly onto a Sephadex G-10 column (2.5 cm×8 cm) andeluted with water. Lyophilization of the appropriate fractions gave thedesired Moraxella serotype C11 decasaccharide 37 (25 mg).

Synthesis of Serotype C11 Decamer Conjugates 38, 39 (FIG. 11B)

First, a conjugation stock solution of decasaccharide 37 was prepared bydissolving the decasaccharide 37 (6.7 mg, 3.82 μmol) in water (0.5 mL).Hydrazine in water (520 μL, 0.05 M, 60 μL) was added to the reactionmixture, was stirred at room temperature for 1 hour and concentrated invacuo. The residue was co-evaporated with water (3×1 mL) and redissolvedin water (300 μL). A solution of tris(2-carboxyethyl)phosphine (TCEP) inwater (39 μL, 0.05 M, 1.95 μmol) was added and stirred for 1 hour.Imject® Conjugation Buffer (Pierce, 300 μL) was added to provide a stocksolution for conjugation to KLH and BSA.

To synthesize the KLH conjugate 38, the conjugation stock solution ofdecasaccharide thiol 37 (139 μL, 0.86 μmol) was added to a solution ofmaleimide-activated keyhole limpet hemocyanin (Imject® KLH, Pierce,Rockford, Ill.) (5 mg, 0.43 μmol maleimide) in water (0.5 mL) was addedand the resulting solution stirred overnight at room temperature. Thereaction mixture was purified by de-salting on D-Salt P-6000 10 mLcolumn (Pierce, Rockford, Ill.). The column was pre-equilibrated with 30mL of purification buffer (Pierce, Prod. No. 77159), the crude materialloaded onto the column and eluted with purification buffer. 1-mLfractions were collected and analyzed for protein content by absorbanceat 280 nm (A₂₈₀). Fractions containing protein were combined andlyophilized to give the desired serotype C11 decamer-KLH conjugate 38.

To synthesize the BSA conjugate 39, the conjugation stock solution ofdecasaccharide thiol 37 (500 μL, 3.0 μmol) was added to a solution ofmaleimide-activated bovine serum albumin (Imject® BSA, Pierce, Rockford,Ill.) (5 mg, ˜1.5 μmol maleimide) in Imject® Conjugation Buffer (Pierce,300 μL diluted with water, 300 μL) and the resulting solution stirredfor 18 hours at room temperature. The reaction mixture was purified byde-salting on D-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). Thecolumn was pre-equilibrated with 30 mL of purification buffer (Pierce,Prod. No. 77159), the crude material loaded onto the column and elutedwith purification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired serotype C11decamer-BSA conjugate 39.

Example 9 Synthesis of Serotype C11 Heptamer and Conjugates ThereofSynthesis of Serotype C11 Heptamer 52 (FIG. 12)

Heptasaccharide 35 (170 mg, 0.056 mmol; see Example 8) was dissolved indioxane (10 mL). AlBN (40 mg, 0.24 mmol) and thioacetic acid (0.3 mL,4.3 mmol) were added, and the solution purged with nitrogen gas for 15minutes. The reaction mixture heated to 75° C. under an inert atmospherefor 18 hours, then cooled to room temperature, quenched with cyclohexene(0.1 mL) and concentrated in vacuo. The crude residue was purified bysize exclusion chromatography using BioRad SX-1 Bio-Beads (2.5 cm×60 cmcolumn) and toluene as the eluent. Appropriate fractions were combinedto give the desired heptasaccharide thioacetate product (160 mg, 92%).

The heptasaccharide thioacetate (88 mg, 0.028 mmol) was dissolved in DMF(3 mL) and ethanol (1.5 mL), and the solution was purged with nitrogenfor 5 minutes. Benzyl chloride (60 μL, 0.52 mmol) and 1.0 M NaOH (0.2mL) were added, and the reaction mixture was stirred for 1 hour. Themixture was diluted with ethyl acetate (50 mL), washed with water (2×50mL) and brine (2×50 mL), dried over Na₂SO₄, filtered and concentrated toa colorless oil (80 mg), which was used without further purification.

Heptasaccharide benzyl thioether (80 mg, 0.025 mmol) was dissolved indry THF (2 mL). In a flame-dried jacketed flask equipped with a dry icecondenser and cooled to −78° C., NH₃ gas was condensed into the flaskunder an Ar atmosphere until the liquid volume reached approximately 15mL. The heptasaccharide benzyl thioether solution was transferred intothe liquid NH₃. Sodium metal (20 mg, 0.87 mmol) was added under positiveAr gas flow and stirred for 10 minutes. The reaction was quenched at−78° C. by the addition of saturated NH₄Cl solution (0.1 mL) and warmedto room temperature. N₂ was bubbled through the reaction mixture toremove excess NH₃, and then the solution was concentrated to dryness.The crude material was purified by size exclusion chromatography on abed of Sephadex G-10 (2.5 cm×8 cm column) by elution with water. Thefractions containing product were concentrated to yield the crudeheptasaccharide with co-eluting salts (80 mg).

The crude heptasaccharide/salt mixture (80 mg) was dissolved in water(0.8 mL) and methanol (0.4 mL), solid NaHCO₃ (40 mg), and aceticanhydride (40 μL) were added. The reaction mixture was stirred for 2hours then loaded directly onto a Sephadex G-10 column (2.5 cm×8 cm) andeluted with water. Lyophilization of the appropriate fractions gave thedesired Moraxella serotype C11 heptasaccharide fragment 52 (15.4 mg).

Synthesis of Serotype C11 Heptamer Conjugates 53, 42 (FIG. 12)

First, a conjugation stock solution of heptasaccharide 40 was preparedas follows. The heptasaccharide 40 (4.9 mg, 3.86 μmol) was dissolved inwater (0.6 mL). Hydrazine in water (0.05 M, 60 μL) was added to thereaction mixture, was stirred at room temperature for 1 hour andconcentrated in vacuo. The residue was co-evaporated with water (3×1 mL)and redissolved in water (300 μL). A solution oftris(2-carboxyethyl)phosphine (TCEP) in water (39 μL, 0.05 M, 1.95 μmol)was added and stirred for 1 hour. Imject® Conjugation Buffer (Pierce,300 μL) was added to provide a stock solution for conjugation to KLH andBSA.

To synthesize the KLH conjugate 53, conjugation stock solution ofheptasaccharide thiol 40 (140 μL, 0.87 μmol) was added to a solution ofmaleimide-activated keyhole limpet hemocyanin (Imject® KLH, Pierce,Rockford, Ill.) (5 mg, 0.43 μmol maleimide) in water (0.5 mL) was addedand the resulting solution stirred overnight at room temperature. Thereaction mixture was purified by de-salting on D-Salt P-6000 10 mLcolumn (Pierce, Rockford, Ill.). The column was pre-equilibrated with 30mL of purification buffer (Pierce, Prod. No. 77159), the crude materialloaded onto the column and eluted with purification buffer. 1-mLfractions were collected and analyzed for protein content by absorbanceat 280 nm (A₂₈₀). Fractions containing protein were combined andlyophilized to give the desired serotype C11 heptamer fragment-KLHconjugate 53.

To synthesize the BSA conjugate 42, conjugation stock solution ofheptasaccharide thiol 40 (500 μL, 3.0 μmol) was added to a solution ofmaleimide-activated bovine serum albumin (Imject® BSA, Pierce, Rockford,Ill.) (5 mg, ˜1.5 μmol maleimide) in Imject® Conjugation Buffer (Pierce,250 μL diluted with water, 250 μL) and the resulting solution stirredfor 18 hours at room temperature. The reaction mixture was purified byde-salting on D-Salt P-6000 10 mL column (Pierce, Rockford, Ill.). Thecolumn was pre-equilibrated with 30 mL of purification buffer (Pierce,Prod. No. 77159), the crude material loaded onto the column and elutedwith purification buffer. 1-mL fractions were collected and analyzed forprotein content by absorbance at 280 nm (A₂₈₀). Fractions containingprotein were combined and lyophilized to give the desired serotype C11heptamer fragment-BSA conjugate 42.

Table 3 provides supporting characterization data for selectedoligosaccharides and intermediates described in Examples 2-9.

TABLE 3 Characterization data compound NMR Mass Spec Series Description# ¹H ¹³C Theo. MALDI 7mer OBn/Allyl 45 √ √ 3175 3195 (M + Na⁺) CoreOBn/SAc √ 3251 OH/SH 46 √ √ 1227 1251 (M + Na⁺) Tetra OBn/Allyl 4 √ √1698 1721 (M + Na⁺) Core OBn/SAc √ √ 1774 1796 (M + Na⁺) OH/SH 5 √ √ 740  763 (M + Na⁺) A OBn/Allyl/N₃ 26 √ √ 3543 OBn/SAc/N₃ √ 3619OBn/SBn/N₃ √ 3664 3665 (M + H⁺) OH/SH/NH₂ √ √ 1388 1411 (M + Na⁺)OH/SH/NHAc 27 √ 1430 1496 (M + Ac) B7 OBn/Allyl 7 √ √ 2743 2764 (M +Na⁺) OBn/SAc √ 2819 2841 (M + Na⁺) OH/SH 8 √ √ 1065 1090 (M + Na⁺) B9OBn/Allyl 10 √ 3608 OBn/SAc √ 3684 OH/SH 11 √ √ 1389 1413 (M + Na⁺) B11OBn/Allyl 19 √ √ 4473 OBn/SAc √ 4549 OH/SH 20 √ √ 1713 1737 (M + Na⁺)C11 OBn/Allyl/N₃ 35 √ √ 3020 3043 (M + Na⁺) Hepta OBn/SAc/N₃ √ 3096Fragment OBn/SBn/N₃ √ 3144 3144 (M⁺) OH/SH/NH₂ √ 1226 1249 (M + Na⁺)OH/SH/NHAc 40 √ 1268 1334 (M + Ac) C11 OBn/Allyl/N₃ 36 √ √ 4408 4429(M + Na⁺) OBn/SAc/N₃ √ 4484 OBn/SBn/N₃ √ 4532 4530 (M⁺) OH/SH/NH₂ √ 17121735 (M + Na⁺) OH/SH/NHAc 37 √ 1754 1819 (M + Ac)

In Table 2, protein assays were performed according to the method ofBradford, M. Anal. Biochem. 1976, 72, 248. Carbohydrate analysis wasperformed according to the method of Dubois, M. et al Anal. Chem. 1956,28, 350. Maldi analysis was performed using 2,5-dihydroxybenzoic acid asa matrix. Copy numbers were determined by the formula: copynumber=[Maldi_((observed))−76,000_((Maldi of BSA alone))]/Antigen MW.Carbohydrate content in KLH sample extrapolated from BSA using theformula: KLH carbohydrate content=BSA carbohydrate content/2.65.

Table 4 provides supporting characterization data for the antigenconjugates described in Examples 2-9.

TABLE 4 characterization data Protein Assay Carb. BSA KLH Assay Avg. %Carbohydrate Conjugate (mg/ (mg/ Hexose Antigen Copy Carb. # DescriptionmL) mL) (mg/mL) MALDI MW No. MALDI Assay Sample 50 Tetra Core-BSA 1.110.33 83,540 740 10.2  9% 30% 49 Tetra Core-KLH 1.34 0.12 740  9% 3% 41B7-BSA 0.91 0.5 89,500 1065 12.7 15% 55% 9 B7-KLH 1.43 0.13 1065  9% 6%48 7mer Core-BSA 1.16 0.35 88,480 1226 10.2 14% 30% 47 7mer Core-KLH1.19 0.1 1226  8% 5% 40 B9-BSA 0.98 0.36 89,460 1389  9.7 15% 37% 12B9-KLH 1.19 0.12 1389 10% 6% 22 B11-BSA 1.09 0.59 102,600 1715 15.5 26%54% 21 B11-KLH 1.14 0.14 1715 12% 10%  29 A-BSA 1.55 0.6 94,309 143012.8 19% 39% 28 A-KLH 1.36 0.19 1430 14% 7% 42 C11 Hepta Core 1.4  0.4593,487 1267 13.8 19% 32% (Trunc C)-BSA 53 C11 Hepta Core 1.2  0.12 126710% 7% (Trunc C)-KLH 39 C11-BSA 1.52 0.47 91,750 1753  9.0 17% 31% 38C11-KLH 1.2  0.16 1753 13% 6%

In Table 5, protein assays, Maldi analysis, copy numbers, andcarbohydrate content were determined as described above with referenceto Table 4.

Example 10 Serum Antibody Production and Purification

Antisera to each antigen-KLH conjugate were raised in New Zealand whiterabbits by four subcutaneous injections of antigen-KLH conjugate over 13weeks. A pre-immune bleed generated 5 mL of baseline serum from eachrabbit. The prime injection (10 μg antigen equivalent) was given as anemulsion in complete Freund's adjuvant (CFA). Subsequent injections (5μg antigen equivalent) were given at three week intervals in incompleteFreund's adjuvant (IFA). Rabbits were bled every two weeks commencingone week after the third immunization. Approximately 25-30 mL of serumper rabbit was generated for each bleeding event, and was aliquoted into1-mL aliquots and frozen at −80° C. Serum was analyzed by ELISA againstthe corresponding antigen-BSA conjugate as described in Example 11.Affinity purification of antisera was conducted with serum from thethird bleed from each rabbit.

Affinity purification of antisera was conducted with serum from thethird bleed from each rabbit. Affinity purification was carried out bycoupling of antigen-BSA conjugates to CNBr-activated Sepharose 4B.Briefly, CNBr-activated Sepharose 4B (0.8 g, 2.5 ml of final gel volume)was washed and re-swelled on a sintered glass filter with 1 mM HCl, thencoupling buffer (0.1M NaHCO₃, 0.25M NaCl, pH 8.5). Antigen-BSA conjugate(1 mg) was dissolved in coupling buffer, mixed with the gel suspensionand incubated overnight at 40° C. Unreacted active groups were cappedwith glycine buffer (0.2M, pH 8.1) and excess adsorbed conjugated waswashed away with coupling buffer, then acetate buffer (0.1 M containing0.5M NaCl, pH 4.3). The column was equilibrated with phosphate bufferedsaline (PBS), pH7.7.

Antisera were affinity purified by diluting clear antiserum (5 mL) 1:1with PBS pH7.7 and applying the diluted antisera to the affinity columnat the rate of 0.3 ml/min and absorbance of eluate was monitored at 280nm. Unbound material (flow through) was collected and analyzed by ELISAusing the general ELISA procedure. The column was washed with PBS untilA280 reached baseline. Bound antibodies were eluted with 0.2M glycine(pH 1.85) into one fraction until the A280 returned to baseline.Fractions were neutralized with 1M Tris-HCl, pH 8.5 immediately aftercollection and the OD at 280 nm was determined. ELISA analysis wasconducted using the corresponding antigen-BSA conjugate according to thegeneral ELISA protocol in Example 11 to confirm the recovered antibodyand the removal of all the antibodies from the original serum. Antibodyquantification was determined by A280 reading of the antibody (a smallamount was diluted to give an OD value of about 1.0) and this value wasdivided by the extinction coefficient of IgG, 1.4, to give mg/mL. Thesolutions were concentrated to ˜1-2 mg/mL, dialyzed against PBS with0.02% sodium azide, aliquoted and frozen at −80° C.

Example 11 ELISA Preparation and Protocol

An oligosaccharide-BSA conjugate solution was prepared by dissolving theconjugate in carbonate buffer (1.59 g Na₂CO₃, 2.93 g NaHCO₃, 0.20 gNaN₃, dissolved and diluted to 1 L in H₂O, final pH 9.5) at aconcentration of 5-10 μg/mL. COSTAR flat bottom EIA 8-well strips wereincubated with oligosaccharide-BSA conjugate solution (100 μL per well)for 24 hours in a humidity chamber to coat the well surfaces. Coatingsolution was removed, each well was rinsed twice with water, and driedon a paper towel. Blocking solution (0.1% BSA in PBS with 0.02%thimerosal, 200 μL) was added to each well and incubated for 2 hours ina humidity chamber. Blocking solution was removed; each well was rinsedwith water and dried on a paper towel.

Serum samples were prepared by 1:5 serial dilutions starting from a1:1,000 dilution of serum in 0.1% BSA in PBS with 0.02% thimerosal.Diluted serum (100 μL) was added to each well and incubated for 2 hoursat room temperature in a humidity chamber. The serum solution wasremoved, the wells were rinsed twice with water and dried on a papertowel. Goat anti-rabbit-HRP conjugate solution (100 μL/well) was addedand incubated for 2 hours at room temperature in a humidity chamber. TheHRP-conjugate solution was removed, and wells were washed three timeswith PBS/0.02% thimerosal/0.05% tween-20, twice with water, and dried ona paper towel. TMB solution (100 μL/well) was added and developed atroom temperature. The reaction was stopped by the addition of 1N HCl(100 μL/well) and the wells were read immediately at A450 (absorbance at450 nm). The titer of the test serum was designated as the dilutionwhich gave an optical density (OD₄₅₀) reading of 0.1 above background.

Example 12 Rabbit Immunogenicity of Synthetic Antigen-KLH Conjugates byELISA

FIGS. 13A-13H depict ELISA results showing IgG IgG antibody titers as afunction of antibody-antigen complex absorption (OD₄₅₀) at 3 serumdilutions of immune sera obtained from 3 succesive bleeds(pre-imune′^(1st) bleed, and final bleed) in rabbits (n=2) immunizedwith antigen-KLH conjugates corresponding to (A) Serotype A-KLH 28; (B)Serotype B7-KLH 9; (C) Serotype B9-KLH 12; (D) Serotype B11-KLH 21; (E)Serotype C11-decamer-KLH 38; (F) Serotype C11-heptamer-KLH 53; (G)heptamer core-KLH 47; and (H) tetramer core-KLH 49. In each case, theantisera were incubated on ELISA plates adsorbed with theircorresponding BSA conjugate, specifically (A) Serotype A-BSA 29; (B)Serotype B7-BSA 41; (C) Serotype B9-BSA 40; (D) Serotype B11-decamer-BSA22; (E) Serotype C11-decamer-BSA 39; (F) Serotype C11-heptamer-BSA 42;(G) heptamer core-BSA 48; and (H) tetramer core-BSA 50 as described inthe ELISA protocol above (Example 11).

FIGS. 13A-13H show that each antiserum was able to identify itscorresponding BSA conjugate, thereby reflecting Ag-selective immuneresponses.

Example 13 Specificity and Cross-Reactivity of Antisera to DifferentAntigens by ELISA

FIGS. 14A-14D depict specificity and cross-reactivity of antisera todifferent synthetic Moraxella LOS oligosaccharides. Antisera fromrabbits immunized with the indicated antigen-KLH conjugatescorresponding to (left to right) KLH alone, tetramer core-KLH 49,hexamer core (B7)-KLH 9, heptamer core-KLH 47, Serotype B11-KLH 21,Serotype A-KLH 28, and Serotype C11-decamer-KLH 38 were incubated withELISA plates adsorbed with antigen-BSA conjugates, including (A)Serotype A-BSA 29; (B) Serotype B11-BSA 22; (C) Serotype C11-decamer-BSA39; and (D) Serotype C11-heptamer fragment-BSA 48. The IgG titers aremeasured a function of antibody-antigen complex absorption (OD₄₅₀) atthe indicated serum dilutions. The data reflect average OD₄₅₀ valuesmeasured from sera from two rabbits, whereby total OD₄₅₀ is measured bysubtracting the background OD₄₅₀ measured from KLH alone.

FIGS. 14A-14D show significant cross-reactivity for the core structuresagainst the full length structures, indicating the potential for singlevalent vaccine(s) effective against all serotypes.

Example 14 Protocol Summary—M. catarrhalis Serum Bactericidal Assay(SBA)

The following procedure was performed to determine the neutralizing ofactivity of serum against M. catarrhalis. Serum was sterile filteredprior to assay and confirmed to be free of contaminates. Serum sampleswere serially diluted 1:5 by adding 20 ul serum to 80 μl PBS containing0.1% gelatin. 50 μl of undiluted, 1:5 dilution, 1:25, and 1:125 dilutionof immune serum was added to 96 well plates. 50 μl of undiluted and 1:5dilution pre-immune serum was added to 96 well plates. 0.25 μg/mlCiprofloxacin was used as a positive control.

Samples and controls were tested in quadruplicate. 20 μl Rabbit serum(Sigma) as a complement source at a 1:8 dilution in PBS was added toeach well. A bacterial suspension of M. catarrhalis (Type A, ATCC 25238;Type B, CCUG 26937 or Type C, CCUG 26404) made so that the suspensionequal to ˜0.100 absorbance which is ˜1.6 10⁸ CFU/mL. The suspension wasdiluted 1:100 to equal 1.6×10⁶ CFU/ml (add 20 μl bacteria to 1.980 mLBHI media). The suspension was diluted 1:100 again (add 100 μl 1:100dilution to 9.9 mL BHI media), then add 30 μl per well for 5×10²CFU/well.

30 μl of M. catarrhalis was added to each well at a density of 500CFU/well. 96 well plates were incubated at 37° C. with 5% CO₂ for 1hour. 50 μl of each well of the 96 well plate was plated onto chocolateagar. Chocolate agar plates were incubated at 37° C. with 5% CO₂overnight. Colonies were counted and recorded.

TABLE 6 Serotype A bactericidal activity data (ATCC 25238) Pre- immuneAverage Immune Average Serum Dilution CFU/well Serum Dilution CFU/well %Killing KLH Carrier no dilution 211 KLH Carrier no dilution 313.5 0.01:5 216 1:5 163.5 22.5 1:25 239.5 0.0 1:125 338.5 0.0 TetraCore nodilution 493 TetraCore no dilution 14 97.2 (49) 1:5 310.5 (49) 1:5 28342.6 1:25 236 52.1 1:125 345 30.0 7mer Core no dilution 394 7mer Core nodilution 3.5 99.1 (47) 1:5 486.5 (47) 1:5 79.5 79.8 1:25 393 0.3 1:125194.5 50.6 ST A (28) no dilution 254.5 ST A (28) no dilution 0 100.0 1:5270.5 1:5 0 100.0 1:25 3.5 98.6 1:125 74 70.9 ST B11 (21) no dilution501.5 ST B11 (21) no dilution 0.5 99.9 1:5 332 1:5 13.5 97.3 1:25 21457.3 1:125 249 50.3 Cipro 0.25 μg/ml 79.5 M. no 368 catarrhalistreatment

TABLE 7 Serotype A bacteridical activity data-(ATCC 25238) Pre ImmuneComparative Average Immune Average % Serum Basis CFU/well Serum DilutionCFU/well Killing KLH Neg Control 298 KLH 1:5 220  26% Carrier Carrier1:20 268  10% 1:80 295  1% 1:160 294  1% Tetra Neg Control 287 Tetra 1:52.25  99% Core Core 1:20 253  12% (49) (49) 1:80 226  21% 1:160 213  26%7mer Neg Control 287 7mer 1:5 1.5  99% Core Core 1:20 230  20% (47) (47)1:80 224  22% 1:160 280  2% ST A Neg Control 287 ST A 1:5 0.25 100% (28)(28) 1:20 32  89% 1:80 103  64% 1:160 184  36% ST B11 Neg Control 173 STB11 1:5 0 100% (21) (21) 1:20 116  33% 1:80 145  16% 1:160 143  17% STB7 Neg Control 154 ST B7 1:5 17  89% (9) (9) 1:20 130  16% 1:80 167  −8%1:160 152  1% ST B9 Neg Control 154 ST B9 1:5 0.5 100% (12) (12) 1:20125  19% 1:80 187 −21% 1:160 133  14% C 11 Neg Control 284 C 11 1:5 0100% Hepta Hepta 1:20 298  −5% Core Core 1:80 302  −6% (53) (53) 1:160285  0% ST C Neg Control 284 ST C 1:5 4.5  98% (38) (38) 1:20 6  98%1:80 357 −26% 1:160 350 −23%

TABLE 8 Serotype B bactericidal activity data (CCUG 26397) PreImmuneComparative Average Immune Average % Serum Basis CFU/well Serum DilutionCFU/well Killing KLH Carrier Neg Control 304 KLH Carrier 1:5 170  44%1:20 299  2% 1:80 174  43% 1:160 273  10% Tetra Core (49) Neg Control304 Tetra Core (49) 1:5 87  71% 1:20 290  5% 1:80 302  1% 1:160 294  3%7mer Core (47) Neg Control 650 7mer Core (47) 1:5 177  73% 1:20 650  0%1:80 650  0% 1:160 650  0% ST A (28) Neg Control 650 ST A (28) 1:5 89 86% 1:20 650  0% 1:80 650  0% 1:160 650  0% ST B11 (21) Neg Control 337ST B11 (21) 1:5 60  82% 1:20 304  10% 1:80 364  −8% 1:160 261  23% ST B7(9) Neg Control 263 ST B7 (9) 1:5 5  98% 1:20 277  −5% 1:80 264  0%1:160 127  52% ST B9 (12) Neg Control 421 ST B9 (12) 1:5 0 100% 1:20 136 68% 1:80 299  29% 1:160 351  17% C 11 Neg Control 421 C 11 1:5 3  99%HeptaCore (53) HeptaCore (53) 1:20 317  25% 1:80 323  23% 1:160 363  14%ST C (38) Neg Control 421 ST C (38) 1:5 5  99% 1:20 346  18% 1:80 295 30% 1:160 303  28%

TABLE 9 Serotype C Killing-Moraxella Catarrhalis CCUG 26404 PreImmuneComparative Average Immune Average % Serum Basis CFU/well Serum DilutionCFU/well Killing KLH Carrier Neg Control 263 KLH Carrier 1:5 146 44%1:20 243  8% 1:80 227 14% 1:160 282 −7% Tetra Core (49) Neg Control 263Tetra Core (49) 1:5 46 83% 1:20 207 21% 1:80 161 39% 1:160 213 19% 7merCore (47) Neg Control 650 7mer Core (47) 1:5 18 97% 1:20 650  0% 1:80650  0% 1:160 650  0% ST A (28) Neg Control 650 ST A (28) 1:5 3.5 99%1:20 650  0% 1:80 650  0% 1:160 650  0% ST B11 (21) Neg Control 650 STB11 (21) 1:5 4 99% 1:20 650  0% 1:80 650  0% 1:160 650  0% ST B7 (9) NegControl 650 ST B7 (9) 1:5 53 92% 1:20 650  0% 1:80 650  0% 1:160 650  0%ST B9 (12) Neg Control 428 ST B9 (12) 1:5 97 77% 1:20 337 21% 1:80 26239% 1:160 451 −5% C 11 Neg Control 428 C 11 1:5 7 98% HeptaCore (53)HeptaCore (53) 1:20 209 51% 1:80 309 28% 1:160 319 25% ST C (38) NegControl 428 ST C (38) 1:5 3 99% 1:20 196 54% 1:80 487 −14%  1:160 33821%

It is intended that the foregoing detailed description be regarded asillustrative rather than limiting, and that it be understood that it isthe following claims, including all equivalents, that are intended todefine the spirit and scope of this invention.

1. A synthetic oligosaccharide 1a or 1b:

where each of R¹ and R² is independently H, a monosaccharide or aoligosaccharide, X is H or a protecting group, L is a linker, and Y is Hor a carrier.
 2. The oligosaccharide of claim 1, which is 1b.
 3. Theoligosaccharide of claim 1, where R¹ is H.
 4. The oligosaccharide ofclaim 1, where R² is H.
 5. The oligosaccharide of claim 1, where atleast one of R¹ or R² is a mono-, di-, tri- or tetra-saccharide.
 6. Theoligosaccharide of claim 1, where at least one of R¹ and R² is amonosaccharide.
 7. The oligosaccharide of claim 1, where each of R¹ andR² is independently a monosaccharide selected from the group consistingof Glc, Gal, and GlcNAc.
 8. The oligosaccharide of claim 1, where theoligosaccharide comprises monosaccharide units selected from the groupconsisting of Glc, Gal, GlcNAc, and Neu5Ac.
 9. The oligosaccharide ofclaim 1, where R¹ is α-Glc-(1→2).
 10. The oligosaccharide of claim 1,where R¹ is β-Gal-(1→4)-α-Glc.
 11. The oligosaccharide of claim 1, whereR¹ is α-Gal-(1→4)-β-Gal-(1→4)-α-Glc.
 12. The oligosaccharide of claim 1,where R¹ is H and R² is H.
 13. The oligosaccharide of claim 1, where R¹is H and R² is α-Glc(1→2).
 14. The oligosaccharide of claim 1, where R¹is H and R² is β-Gal-(1→4)-α-GlcNAc.
 15. The oligosaccharide of claim 1,where R¹ is H and R² is α-Gal-(1→4)-β-Gal-(1→4)-α-GlcNAc.
 16. Theoligosaccharide of claim 1, where R¹ is H and R² isβ-Gal-(1→4)-α-Glc(1→2).
 17. The oligosaccharide of claim 1, where R¹ isH and R² is α-Gal-(1→4)-β-Gal-(1→4)-α-Glc(1→2).
 18. The oligosaccharideof claim 1, where R¹ is H and R² is α-GlcNAc(1→2).
 19. Theoligosaccharide of claim 1, where R¹ is α-Glc-(1→2) and R² is H.
 20. Theoligosaccharide of claim 1, where R¹ is α-Glc-(1→2) and R² is α-GlcNAc.21. The oligosaccharide of claim 1, where R¹ is α-Glc-(1→2) and R² isα-Glc-(1→2).
 22. The oligosaccharide of claim 1, where R¹ is α-Glc-(1→2)and R² is β-Gal-(1→4)-α-Glc.
 23. The oligosaccharide of claim 1, whereR¹ is α-Glc-(1→2) and R² is β-Gal-(1→4)-α-GlcNAc.
 24. Theoligosaccharide of claim 1, where R¹ is α-Glc-(1→2) and R² isα-Gal(1→4)-β-Gal(1→4)-α-Glc(1→2).
 25. The oligosaccharide of claim 1,where R¹ is α-Glc-(1→2) and R² is α-Gal(1→4)-β-Gal(1→4)-α-GlcNAc(1→2).26. The oligosaccharide of claim 1, where R¹ is β-Gal-(1→4)-α-Glc and R²is H.
 27. The oligosaccharide of claim 1, where R¹ is β-Gal-(1→4)-α-Glcand R² is α-Glc(1→2).
 28. The oligosaccharide of claim 1, where R¹ isβ-Gal-(1→4)-α-Glc and R² is β-Gal-(1→4)-α-Glc(1→2).
 29. Theoligosaccharide of claim 1, where R¹ is β-Gal-(1→4)-α-Glc and R² isα-Gal-(1→4)-β-Gal-(1→4)-α-Glc(1→2).
 30. The oligosaccharide of claim 1,where R¹ is β-Gal-(1→4)-α-Glc and R² is α-GlcNAc(1→2).
 31. Theoligosaccharide of claim 1, where R¹ is β-Gal-(1→4)-α-Glc and R² isβ-Gal-(1→4)-α-GlcNAc(1→2).
 32. The oligosaccharide of claim 1, where R¹is β-Gal-(1→4)-α-Glc and R² is α-Gal-(1→4)-β-Gal-(1→4)-α-GlcNAc (1→2).33. The oligosaccharide of claim 1, where R¹ isα-Gal-(1→4)-β-Gal-(1→4)-α-Glc and R² is H.
 34. The oligosaccharide ofclaim 1, where R¹ is α-Gal-(1→4)-β-Gal-(1→4)-α-Glc and R² is α-Glc(1→2).35. The oligosaccharide of claim 1, where R¹ isα-Gal-(1→4)-β-Gal-(1→4)-α-Glc and R² is α-GlcNAc.
 36. Theoligosaccharide of claim 1, where R¹ is α-Gal-(1→4)-β-Gal-(1→4)-α-Glcand R² is β-Gal-(1→4)-α-Glc.
 37. The oligosaccharide of claim 1, whereR¹ is α-Gal-(1→4)-β-Gal-(1→4)-α-Glc and R² is β-Gal-(1→4)-α-GlcNAc. 38.The oligosaccharide of claim 1, where R¹ isα-Gal-(1→4)-β-Gal-(1→4)-α-Glc and R² is α-Gal-(1→4)-β-Gal-(1→4)-α-Glc.39. The oligosaccharide of claim 1, where R¹ isα-Gal-(1→4)-β-Gal-(1→4)-α-Glc and R² isα-Gal-(1→4)-β-Gal-(1→4)-α-GlcNAc.
 40. The oligosaccharide of any one ofclaims 1 to 39, where L is an alkylene thiol linker.
 41. The syntheticoligosaccharide of any one of claims 1 to 40, where Y is a carrierselected from the group consisting of proteins, peptides, lipids,polymers, dendrimers, virosomes, and virus-like particles or combinationthereof.
 42. The synthetic oligosaccharide of claim 41, where thecarrier is a carrier protein.
 43. The synthetic oligosaccharide of claim42, where the carrier protein is selected from the group consisting ofbacterial toxoids, toxins, exotoxins, and nontoxic derivatives thereof.44. The synthetic oligosaccharide of claim 43, wherein the carrierprotein is selected from the group consisting of tetanus toxoid, tetanustoxin Fragment C, diphtheria toxoid, CRM, cholera toxoid, Staphylococcusaureus exotoxins or toxoids, Escherichia coli heat labile enterotoxin,Pseudomonas aeruginosa exotoxin A, genetically detoxified variantsthereof; bacterial outer membrane proteins, serotype B outer membraneprotein complex (OMPC), outer membrane class 3 porin (rPorB), porins;keyhole limpet hemocyanine (KLH), hepatitis B virus core protein,thyroglobulin, albumins, and ovalbumin; pneumococcal surface protein A(PspA), pneumococcal adhesin protein (PsaA); purified protein derivativeof tuberculin (PPD); transferrin binding proteins, peptidyl agonists ofTLR-5; and derivatives and/or combinations of the above carriers. 45.The synthetic oligosaccharide of claim 44, wherein the carrier proteinis selected from the group consisting of CRM 197, Neisseriameningitidis, bovine serum albumin (BSA), human serum albumin (HSA),poly(lysine:glutamic acid), flagellin of motile bacteria, andderivatives and/or combinations thereof.
 46. The syntheticoligosaccharide of claim 44, wherein the carrier protein is selectedfrom the group consisting of tetanus toxoid, CRM 197, and OMPC.
 47. Apharmaceutical composition comprising a least one oligosaccharide of anyone of claims 1 to 46 in an effective amount to stimulate an immuneresponse, optionally further comprising a pharmaceutically acceptablecarrier.
 48. The pharmaceutical composition of claim 47, furthercomprising an adjuvant.
 49. The pharmaceutical composition of any one ofclaim 47 or 48 wherein the immune response is an antigen-specific immuneresponse.
 50. A composition comprising a synthetic oligosaccharide ofany one of claims 1 to 46 and a pharmaceutically acceptable vehicle. 51.The composition of claim 50, comprising a plurality of differentoligosaccharides, where each oligosaccharide is an antigen 1b.
 52. Acomposition comprising at least two oligosaccharides, wherein a first isa M. catarrhalis serotype C antigen and a second is a M. catarrhaliscore antigen.
 53. The composition of claim 52, further comprising athird oligosaccharide which is a M. catarrhalis serotype A antigen. 54.The composition of claim 52, further comprising a fourth oligosaccharidewhich is a M. catarrhalis serotype B antigen.
 55. The composition ofclaim 54, further comprising a fourth oligosaccharide which is a M.catarrhalis serotype B antigen.
 56. A composition comprising at leasttwo oligosaccharides, wherein a first is a M. catarrhalis serotype Bantigen and a second is a M. catarrhalis core antigen.
 57. Thecomposition of claim 56, further comprising a third oligosaccharidewhich is a M. catarrhalis serotype A antigen.
 58. A compositioncomprising at least two oligosaccharides, wherein a first is a M.catarrhalis serotype A antigen and a second is a M. catarrhalis coreantigen.
 59. A composition comprising at least two oligosaccharides,wherein a first is a M. catarrhalis serotype B antigen and a second is aM. catarrhalis serotype C antigen.
 60. The composition of claim 59,further comprising a third oligosaccharide which is a M. catarrhalisserotype A antigen.
 61. A composition comprising at least twooligosaccharides, wherein a first is a M. catarrhalis serotype A antigenand a second is a M. catarrhalis serotype C antigen.
 62. A compositioncomprising at least two oligosaccharides, wherein a first is a M.catarrhalis serotype A antigen and a second is a M. catarrhalis serotypeB antigen.
 63. The composition of any of claims 51 to 62, furthercomprising an adjuvant.
 64. The composition of claim 63, where theadjuvant is selected from the group consisting of aluminum salts, RIBI,toll-like receptor agonists, AS01 AS02 AS03, AS04, AS05,CpG-oligodeoxynucleotide, MF-59, Montanide ISA-51 VG, Montanide ISA-720,Quil A, QS21, synthetic saponins, immunostimulating complexes, stearyltyrosine, virus-like particles, reconstituted influenza virosomes,cytokines, mast cell activator compound 48/80, liposomes, muramyldipeptides, SAF-1, and combinations thereof.
 65. The composition of anyof claims 51 to 62, comprising an amount of at least one oligosaccharidesufficient to confer immunity against Moraxella.
 66. A compositioncomprising an oligosaccharide of any one of claims 1 to 46 as a vaccine.67. An antibody preparation against an oligosaccharide according to anyone of claims 1 to
 41. 68. The antibody preparation of claim 67, wherethe antibody preparation comprises at least one member from the groupconsisting of polyclonal antibody, monoclonal antibody, mouse monoclonalIgG antibody, humanized antibody, chimeric antibody, single chainantibodies, fragment thereof, or combination thereof.
 69. A method oftreating a disease associated with M. catarrhalis infection, comprisingadministering effective amount for inducing an immune response againstMoraxella of an oligosaccharide of any of claims 1 to 46 or antibodythereto.
 70. A method of treating a disease associated with M.catarrhalis infection, comprising administering to a patient in needthereof a composition of any of claims 1 to
 46. 71. The method of claim69 or 70, wherein the disease is COPD.
 72. The method of claim 69 or 70,wherein the disease is AOM.
 73. The method of claim 69 or 70, whereinthe patient is human.
 74. A method for producing antibodies comprising:(a) administering to a subject an effective amount of at least oneoligosaccharide of any one of claims 1 to 41, for producing antibodiesspecific for Moraxella; optionally further comprising an adjuvant. (b)isolating antibodies from the subject.
 75. A method for producingmonoclonal antibodies comprising: (a) administering to a subject aneffective amount of at least one oligosaccharide of any one of claims 1to 41, for producing antibodies specific Moraxella; (b) isolatingantibodies from the subject. (c) fusing antibody producing cells fromthe subject to myeloma cells, and (d) harvesting antibodies producedfrom a fusion subclone.
 76. The method of claims 74 and 75, wherein thesubject is a rabbit.
 77. The method of claims 74 and 75, wherein thesubject is a human.
 78. An antibody producing cell obtainable byperforming steps (a) to (c) of claim
 75. 79. An antibody obtainable byperforming steps (a) to (d) of claim
 75. 80. A method of diagnosing thepresence of Moraxella in a sample, comprising contacting the sample withan antibody of claim 67, 68, 78, or
 79. 81. A compound selected from thegroup consisting of:


82. A compound of the formula:

where R is allyl or R═(CH₂)₃Sac.
 83. A compound of the formula:


84. A compound of the formula:


85. A compound of the formula:


86. A compound of the formula:


87. A compound of the formula:


88. A compound of the formula:


89. A compound of the formula:


90. A compound of the formula:


91. A compound of the formula:

where Y is a carrier.
 92. The compound of claim 91, where the carrier isa carrier protein.
 93. The compound of claim 92, where the carrierprotein is selected from the group consisting of bacterial toxoids,toxins, exotoxins, and nontoxic derivatives thereof.
 94. The compound ofclaim 92, wherein the carrier protein is selected from the groupconsisting of tetanus toxoid, tetanus toxin Fragment C, diphtheriatoxoid, CRM, cholera toxoid, Staphylococcus aureus exotoxins or toxoids,Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosaexotoxin A, genetically detoxified variants thereof; bacterial outermembrane proteins, serotype B outer membrane protein complex (OMPC),outer membrane class 3 porin (rPorB), porins; keyhole limpet hemocyanine(KLH), hepatitis B virus core protein, thyroglobulin, albumins, andovalbumin; pneumococcal surface protein A (PspA), pneumococcal adhesinprotein (PsaA); purified protein derivative of tuberculin (PPD);transferrin binding proteins, peptidyl agonists of TLR-5; andderivatives and/or combinations of the above carriers.
 95. The compoundof claim 92, wherein the carrier protein is selected from the groupconsisting of CRM 197, Neisseria meningitides, bovine serum albumin(BSA), human serum albumin (HSA), poly(lysine:glutamic acid), flagellinof motile bacteria, and derivatives and/or combinations thereof.
 96. Thecompound of claim 92, wherein the carrier protein is selected from thegroup consisting of tetanus toxoid, CRM 197, and OMPC.
 97. A method ofsynthezing a compound of the formula:

comprising contacting a first intermediate of the formula:

with a second intermediate of the formula:

where R⁷ is a Bn or is a monosaccharide or oligosaccharide; R⁸ is a Bnor is a monosaccharide or oligosaccharide; and R⁹ is a protecting groupor linker consisting of —CH₂CH═CH₂, —CH₂CCH, pentenyl, alkenylene,oligoalkyl thiol.
 98. The method of claim 97, where the compound is: